The highly oligophagous tephritid Chaetostomella cylindrica infests the flower heads of six genera and ten species of thistles in Lebanon. It predominantly utilizes two hosts occurring in sympatry, Notobasis syriaca and Onopordum illyricum. Previous work showed that adult flies emerging from N. syriaca fit more closely the description of the species, particularly with respect to the colour and pattern on the mesonotum; furthermore, significant differences were observed between the aculeus shape and length. This study investigates the biology of the immatures and compares adults from the two host races behaviourally and genetically. Larvae of both races fed in a similar way, with each larva destroying 3-10 achenes; however, the oviposition behaviour of females differed. Females of the Onopordum-associated flies laid an average of three eggs per head, and deposited the eggs glued to each others in a cluster, while females of the Notobasis-associated flies deposited their eggs unattached, usually with one egg per head. Subtle differences were also observed in the post-mating behaviour of adult males. DNA sequencing of an amplified fragment of the mitochondrial NADH-dehydrogenase subunit 1 gene revealed 44 single nucleotide polymorphisms in 622 base pairs. A PCR-RFLP method was developed to distinguish the two host-associated populations. Together with previously published morphometric studies, our data show that C. cylindrica consists of distinct host races, which seem to be reproductively isolated as two separate genetic lineages were observed.