The differential epidemiology of D. repens and D. immitis is still poorly understood due to the lack of a diagnostic method which would make possible the routine identification of these parasites as developing larvae either in the vector or in unsuitable hosts, including man. The PCR-based method here described allows: i) the unambiguous identification of mature and immature adult worms in bioptic material, of microfilariae in blood samples and of developing larvae in mosquito vectors; ii) the analysis of samples stored either dry or in various preservation media, with the exception of formalin. The high specificity and sensitivity of the diagnosis improve the perspectives for comparative epidemiological investigations on D. repens and D. immitis in areas where the two nematodes are sympatric.