The polyphagous fruit fly, Terellia serratulae (Linnaeus) (Diptera: Tephritidae), exploits hosts belonging to three genera of thistles: Carduus Linnaeus, Cirsium Miller, and Picnomon Adanson (Asteraceae). The difference in phenology among its hosts suggests intraspecific variation. Comparative morphometric and genetic studies revealed differences among its populations. Adults reared from different hosts showed intraspecific morphological variations. Canonical discriminant analysis based on two head and four wing measurements divided the adults into four distinct clusters with 70% accuracy, reflecting four host-associated populations. The most useful predictors in distinguishing adults associated with the different host plants were wing width and head length, in addition to ovipositor length for females. Only the ovipositor tip in females reared from Picnomon acarna (Linnaeus) Cassini was clearly distinct. Mitochondrial DNA sequencing revealed genetic differentiation among the different populations of T. serratulae with the P. acarna-associated population being most distinct. Sequencing a region of the mtND1 gene and mtCOXI gene revealed nine and seven haplotypes, respectively. Surprisingly, haplotype sequences of flies emerging from P. acarna showed a sequence divergence of over 3% for both genes. This study provides morphometric and molecular evidence supporting that the Lebanese T. serratulae population associated with P. acarna most likely constitutes a distinct host race.