Genetic studies of Trypanosoma brucei have been mainly based on rodent inoculation (RI) for isolation of trypanosome strains. However, Trypanosoma brucei gambiense is difficult to grow in rodents. The development and use of the Kit for In Vitro Isolation (KIVI) of trypanosomes has led to a better isolation success. However, some authors report a genetic monomorphism in T. b. gambiense, and the extensive use of the KIVI was suspected as being responsible for this low genetic diversity. In the present work, trypanosome stocks were isolated from both humans and pigs in an active sleeping sickness focus in Côte d'Ivoire. Two methods were simultaneously used for this purpose: KIVI and rodent inoculation. None of the human stocks grew in rodents. Some of the stocks originating from pigs could be isolated with both methods. Each of these stocks (from the same pig) showed a different isoenzymatic pattern according to the isolation method used. All the human stocks identified belonged to the major zymodeme 3 of T. b. gambiense group 1, whereas the stocks isolated from pigs belonged to a new group of zymodemes even if they were genetically closely related. These observations may have significant implications when analysing the population structure of T. brucei, and also raise again the question of the importance of the animal reservoir in Human African Trypanosomiasis (HAT).