The Polymerase Chain Reaction (PCR) technique was used for the identification of natural trypanosome infections in Glossina longipalpis (Diptera: Glossinidae) in Côte d'Ivoire. A total number of 139 flies were examined microscopically for the presence of trypanosomes. Out of them 50 were detected positive and were subsequently prepared for the PCR using primers specific for Trypanosoma (Nannomonas) congolense of Savannah, Riverine-Forest, Kilifi, and Tsavo types, T. (N.) simiae, T. (Duttonella) vivax and Trypanozoon. Almost 90% of the infections detected by the PCR were attributed to Nannomonas, especially T. congolense Savannah and Riverine-Forest types, with many infections in which both of these two types were present T. simiae and T. vivax were also detected in some flies. The sequence specificity of the PCR products was confirmed by hybridization with parasite-type specific DNA probes. Differences between parasitological and PCR results are discussed.