This resource contains tsetse fly count data recorded during two intensive surveys in Mambwe District, Eastern Province, Zambia in 2013. Tsetse sampling was conducted along a 60 kilometre transect from the plateau region on the eastern side of the Luangwa Valley to the floor of the valley, near Mfuwe Airport. Tsetse flies were sampled using black screen fly rounds. The first survey was conducted in May and June (cold, dry season) and the second survey in November (hot dry season). The Luangwa Valley in Zambia is an old focus of human sleeping sickness (Trypanosoma brucei rhodesiense) and sporadic outbreaks have occurred in the human population. In recent years there has been an influx of people migrating from the densely populated plateau region resulting in a significant change in land-use in the study area, potentially influencing the distribution and density of tsetse. This work was part of a wider research project, the Dynamic Drivers of Disease in Africa Consortium (DDDAC) and contributed to the Zambia trypanosomiasis case study. The research was funded by NERC project no NE/J000701/1 with support from the Ecosystem Services for Poverty Alleviation Programme (ESPA). Full details about this dataset can be found at https://doi.org/10.5285/59fc6e1a-e1cb-4bcf-8b49-68211b2a363a