Full Text Article. Also available at: https://idpjournal.biomedcentral.com/articles/10.1186/s40249-017-0340-0Background: Changes of land cover modify the characteristics of habitat, host-vector interaction and consequently infection rates of disease causing agents. In this paper, we report variations in tsetse distribution patterns, abundance and infection rates in relation to habitat types and age in the Maasai Steppe of northern Tanzania. In Africa, Tsetse-transmitted trypanosomiasis negatively impacted human life where about 40 million people are at risk of contracting the disease with dramatic socio-economic consequences, for instance, loss of livestock, animal productivity, and manpower. Methods: We trapped tsetse flies in dry and wet seasons between October 2014 and May 2015 in selected habitats across four villages: Emboreet, Loiborsireet, Kimotorok and Oltukai adjacent to protected areas. Data collected include number and species of tsetse flies caught in baited traps, PCR identification of trypanosome species and extraction of monitored Normalized Difference Vegetation Index (NDVI) data from Moderate Resolution Imaging Spectrometer (MODIS). Results: Our findings demonstrate the variation of tsetse fly species abundance and infection rates among habitats in surveyed villages in relation to NDVI and host abundance. Results have shown higher tsetse fly abundance in Acacia-swampy ecotone and riverine habitats for Emboreet and other villages, respectively. Tsetse abundance was inconsistent among habitats in different villages. Emboreet was highly infested with Glossina swynnertoni (68%) in ecotone and swampy habitats followed by G. morsitans (28%) and G. pallidipes (4%) in riverine habitat. In the remaining villages, the dominant tsetse fly species by 95% was G. pallidipes in all habitats. Trypanosoma vivax was the most prevalent species in all infected flies (95%) with few observations of co-infections (with T. congolense or T. brucei). Conclusions: The findings of this study provide a framework to mapping hotspots of tsetse infestation and trypanosomiasis infection and enhance the communities to plan for effective control of trypanosomiasis.