Hearts' hopes of Uefa Cup qualification petered out with defeat at Rugby Park following a penalty from Kilmarnock striker Steven Naismith.
Steven Naismith scored from the penalty spot against Hearts
Roman Bednar and Christophe Berra went close for Hearts early on, while Saulius Mikoliunas squandered a great chance for the Edinburgh side.
Momo Sylla fired just over at the other end, while Aime Koudou forced a good save from goalkeeper Craig Gordon.
Naismith fired home from the spot after a Gordon challenge after 82 minutes.
And the incident involving the two players believed to be the targets for watching Aston Villa manager Martin O'Neill settled the game.
Hearts had needed to win - and hope that Aberdeen failed to beat Rangers - if they were to leapfrog Aberdeen into third place.
But, by then, news that the Dons were beating Rangers ensured that the result in Ayrshire was virtually academic.
Ibrahim Tall and Jose Goncalves had replaced full-backs Christos Karipidis and Takis Fyssas following last week's Edinburgh derby victory.
Kilmarnock were unchanged after their fine win over Rangers.
But it was Hearts who were first to threaten, Roman Bednar flicking the ball across the face of goal after a cross to the near post by Andrew Driver.
Christophe Berra stole in at the back post as the Kilmarnock defence hesitated at a free-kick, but the defender's header from a difficult angle struck the side netting.
Gordon Greer lost the ball to Bednar but made a fine recovery tackle to prevent the striker firing on goal from 16 yards.
Aime Koudou was given a chance to impress against Hearts
Killie had more possession without creating chances and Aime Koudou headed well wide when he found space for himself at a corner.
Mikoliunas missed a glorious chance at the other end when found 12 yards out by a Driver cross but failed to connect properly with his shot.
Killie took heart and a Momo Sylla drive flew just over from the edge of the box, while Koudou forced Gordon into a one-handed parry with a low drive.
Mikoliunas fired just over from 20 yards as Hearts responded, while a last-ditch challenge denied Sylla at the other end.
Kilmarnock boss Jim Jefferies withdrew Greer and Colin Nish at half-time, with Garry Hay and Gary Wales coming on in their place.
Frazer Wright headed straight at Gordon from 12 yards following a corner.
And Killie secured victory when the ball broke off Tall just outside the Hearts box.
Naismith collapsed to the ground as Gordon came out to challenge, with the striker firing the penalty into the corner.
Kilmarnock: Combe, O'Leary, Frazer Wright, Greer (Hay 45), Grant Murray, Fowler, Locke, Sylla, Naismith, Nish (Wales 46), Koudou (Gibson 61).
Subs Not Used: Harpur, Dodds, Di Giacomo, Hamill.
Booked: Frazer Wright, Sylla, Gibson.
Goal: Naismith 82 pen.
Hearts: Gordon, Berra, Goncalves, Zaliukas, Tall, Mikoliunas, Driver, Kingston, Ivaskevicius, Bednar, Pospisil.
Subs Not Used: Banks, Wallace, McCann, Elliot, Neilson, Pilibaitis, Karipidis.
Booked: Ivaskevicius, Berra, Mikoliunas, Gordon.
Ref: W Collum