Michal Kadlec's controversial penalty in the dying seconds at Hampden all but killed off Scotland's hopes of qualifying for the Euro 2012 finals.
Darren Fletcher set up Kenny Miller to fire home for the Scots seconds before the break, but Jaroslav Plasil forced in an equaliser after 78 minutes.
Fletcher rolled in Scotland's second from Miller's pass five minutes later.
But Kadlec fired home after Jan Rezek appeared to dive under Danny Wilson's challenge in stoppage time.
And Craig Levein and his side were left raging moments later when no penalty was given and Christophe Berra was booked instead by Dutch referee Kevin Blom when the defender was clipped in the opposite penalty box.
It leaves the Scots five points adrift of the Czechs with only one game in hand as the two sides chase the runners-up spot in Group I behind leaders Spain.
Levein had gambled by handing starts to captain Fletcher and Alan Hutton despite the pair having had no competitive action this season through illness and injury respectively.
Michal Bilek gave first caps to midfielder Petr Jiracek and Jan Lastuvka, the Dnipro goalkeeper who had loan spells with Fulham and West Ham while with Shakhtar Donetsk being a surprise deputy for the injured Petr Cech ahead of the more experienced Jaroslav Drobny of Hamburg.
Viktoria Pilsen man Jiracek began confidently and, within a couple of minutes, found space behind Hutton to create a great chance for the Czechs.
Collecting Baros's through ball, Jiracek returned it into the path of the Galatasaray striker, who side-footed wildly over from 12 yards.
Former Liverpool and Aston Villa forward Baros created another chance with a curling free-kick that found Jan Rajnoch unmarked, but the Ankaragucu defender flashed a header wide.
The Scots were looking nervous and their mood was not helped by a poor challenge from Scott Brown on Tomas Rosicky that led to a yellow card for the Celtic midfielder, meaning he is suspended for Tuesday's visit by Lithuania.
Charlie Adam was fortunate to get away with a studs-first challenge inside his own penalty box that left Baros writhing rather theatrically in pain.
Scotland worked themselves into the game but were being restricted to half chances.
That all changed a minute before the break when Fletcher, who until then had been suffering an undistinguished return to action, slipped the ball through to Miller and the striker's low drive nestled just inside the far post off the goalkeeper's outstretched foot.
The Czechs showed their intent immediately after the break and, when Rosicky threaded the ball through to Baros, the forward's low drive from the edge of the box slipped a foot wide of the far post.
Goalscorer Miller followed Brown into the suspension list, but the Scots were now beginning to enjoy more quality possession.
Czech defender Roman Hubnik fired into the side netting from close range when the Scots failed to clear their lines, while Miller went close to adding to Scotland's lead when his flick from a Morrison cross to the front post sent the ball spinning across the face of goal.
Scotland lost Phil Bardsley to injury and immediately his replacement, Wilson, was found absent at the back post as a cross found Rezek at the back post and his ball into the six-yard box was deflected home by Plasil.
The Scots were soon back ahead through Fletcher's cool finish from Miller's precise ball, but Kadlec fired a powerful reply from the spot after substitute Rezek went down inside the box.
Levein's side threw everything forward and had strong appeals for a penalty of their own when Berra went down and, although a draw was probably a fair result, the Scots will feel aggrieved by the thrilling finish.
Scotland: McGregor, Hutton, Caldwell, Berra, Bardsley (Wilson 75), Naismith (Robson 86), Fletcher, Adam (Cowie 79), Brown, Morrison, Miller. Subs Not Used: Gilks, Goodwillie, Bannan, Forrest.
Booked: Brown, Miller, Berra, Wilson.
Goals: Miller 44, Fletcher 83.
Czech Republic: Lastuvka, Hubnik, Rajnoch, Sivok, Kadlec, Hubschman, Plasil, Petrzela (Rezek 56), Rosicky ,Jiracek (Pekhart 77), Baros (Vacek 90). Subs Not Used: Drobny, Pudil, Pospech, Lafata.
Booked: Jiracek, Plasil, Rezek, Baros, Pekhart.
Goals: Plasil 78, Kadlec 90 pen.
Referee: Kevin Blom (Netherlands)