Parasitoids of herbivorous insects have frequently evolved specialized lineages exploiting hosts occurring on different plants. This study investigated whether host specialization is also observed when closely related parasitoids exploit herbivorous hosts sharing the same host plant. The question was addressed in economically relevant aphid parasitoids of the Lysiphlebus fabarum group. They exploit two aphid species (Aphis fabae cirsiiacanthoides and Brachycaudus cardui), co-occurring in mixed colonies (syntopy) on the spear thistle (Cirsium vulgare). Two morphologically distinguishable parasitoid lineages of the genus Lysiphlebus were observed and each showed virtually perfect host specialization on one of the two aphid species in this system. From A. f. cirsiiacanthoides, only females emerged that morphologically belonged to Lysiphlebus cardui, while males and females belonging to L. fabarum hatched from B. cardui. Microsatellite analyses indicated clear genetic differentiation of L. fabarum and L. cardui. L. cardui comprised only two distinct asexual lineages, one of which predominated throughout the area investigated. Population genetic analysis of sexual L. fabarum showed evidence for relatively strong spatial structuring and limited dispersal ability. Hyperparasitoids emerged from a large proportion of aphid mummies. One species, Pachyneuron aphidis, was significantly associated with B. cardui/L. fabarum mummies, indicating that host specialization may even extend to the trophic level above parasitoids.