We investigated the dynamics of Glossina spp. and their role in the transmission of trypanosomiasis in the sleeping sickness endemic Serengeti ecosystem, northwestern Tanzania. The study investigated Glossina species composition, trap density, trypanosome infection rates, and the diversity of trypanosomes infecting the species. Tsetse were trapped using monopyramidal traps in the mornings between 06:00 to 11:00 and transported to the veterinary laboratory in Serengeti National Park where they were sorted into species and sex, and dissected microscopically to determine trypanosome infection rates. Age estimation of dissected flies was also conducted concurrently. Tsetse samples positive for trypanosomes were subjected to PCR to determine the identity of the detected trypanosomes. Out of 2,519 tsetse trapped, 1,522 (60.42%) were G. swynnertoni, 993 (39.42%) were G. pallidipes, three (0.12%) were G. m. morsitans, and one (0.04%) was G. brevipalpis. The trap density for G. swynnertoni was between 1.40 and 14.17 while that of G. pallidipes was between 0.23 and 9.70. Out of 677 dissected G. swynnertoni, 63 flies (9.3%) were infected, of which 62 (98.4%) were females. A total of 199 G. pallidipes was also dissected but none was infected. There was no significant difference between the apparent densities of G. swynnertoni compared to that of G. pallidipes (t = 1.42, p = 0.18). Molecular characterization of the 63 infected G. swynnertoni midguts showed that 19 (30.2%) were trypanosomes associated with suid animals while nine (14.3%) were trypanosomes associated with bovid animals and five samples (7.9%) had T. brucei s.l genomic DNA. Thirty (47.6%) tsetse samples could not be identified. Subsequent PCR to differentiate between T. b. brucei and T. b. rhodesiense showed that all five samples that contained the T. brucei s.l genomic DNA were positive for the SRA molecular marker indicating that they were T. b. rhodesiense. These results indicate that G. swynnertoni plays a major role in the transmission of trypaniosomiasis in the area and that deliberate and sustainable control measures should be initiated and scaled up.