Competition and cooperation are well-recognized biological phenomena, even among parasites. Co-infection of parasites in a single host leads to several outcomes, one being competition for a limited resource. Here, the behaviour of mixed infection was evaluated using two isolates of Trypanosoma cruzi, previously typed as belonging to genotypes TcI and TcII. The growth in vitro and in the different compartments of the gut of Triatoma brasiliensis was studied. In vitro growth showed that MDID/BR/1999/M1 (TcI) has a doubling time of 19.5 h and MIDID/BR/1999/JCPD4 (TcII) of 9.6 h, while the mixed infection group presented a doubling time of 13.9 h. In vivo, three groups of infection were done: M1/TcI, JCPD4/TcII and mixed infection (50% of each strain), respectively. All comparisons among the groups were done using the Kruskal–Wallis non-parametric test. The data showed that the in vitro culture of mixed populations has a similar pattern to the growth of M1/TcI, apparently suggesting a positively selection for M1/TcI strain, in axenic culture. In the gut of the insects, M1/TcI isolate and mixed infections colonized predominantly the rectalwall and rectal lumen, in contrast to the JCPD4/TcII isolate, which was found mainly colonizing the small intestine. According to the isolates investigated, it could be concluded that the doubling time was not determinant factor for the final composition of a co-infection. Moreover, mixed infections resulted in a homogenous distribution of the parasites, comparing to the isolates studied separately. Apparently, in the gut of the bugs, the simultaneous presence of JCPD4/TcII isolate resulted in an improvement of the number of parasites from M1/TcI.