This data set includes the results of nested PCR-analysis of tsetse (Glossina sp.) gut samples with primers described by Adams et.al. (2006) and Hamilton et al (2004) from a tsetse (Glossina sp.) collection carried out in Nigeria between 2014 and 2016. Traps were employed for one to three days in four different National Parks, one Game Reserve and two tsetse invested areas. Each tsetse was identified morphologically and dissected in the field. The gut samples were homogenized in the field. DNA was purified from preserved homogenized gut using the Qiagen Blood and Tissue Kit. Presence of trypanosomes was investigated by amplification of Internal Transcribed Spacer-1 (ITS1). Identification of the respective trypanosome species was done based on lengths polymorphism of the ITS1 region by estimating the approximate size of the DNA amplicon based on electrophoretic separation in agarose gels, as well as sequencing of selected ITS1 amplicons and additional glycosomal Glyceraldehyde-3-phosohate dehydrogenase
