The black fly, Simulium aureohirtum was geographically widespread, and encompasses a wide range of ecological conditions. A total of 1303 larvae from 21 sampling sites throughout Thailand were cytologically examined. Fourteen chromosome rearrangements were found and all were paracentric inversions. Most inversions (12 of 14) were present at low frequency and geographically restricted to a particular population, suggesting they originated recently. Significant genetic differentiation between populations were detected (F _ST=0.092, p=0.0002). Detection of an isolation by distance (r=0.497, p=0.004) suggests that gene flow was limited by geographic distance. Inversion frequency of IIL-1 was significantly associated with latitude (r=0.832, p<0.01), altitude (r=0.471, p<0.05) and minimum annual air temperature (r=-0.748, p<0.01) indicating that it might be maintained in populations by selection. The frequency of IIL-2 was significantly associated with altitude (r=0.778, p<0.01) and minimum annual air temperature (r=-0.570, p<0.01) but because it was present at low frequency (<7%), selection probably was not involved. This inversion more likely represents a relict of ancestral polymorphisms. Comparison of the chromosome banding patterns revealed that S. aureohirtum is more closely related to S. ruficorne than to the S. ornatipes complex.