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The origins of extant Glossina pallidipes Austen (Diptera: Glossinidae) populations in the ecologically well-studied Lambwe and Nguruman valleys in Kenya are controversial because populations have recovered after seemingly effective attempts to achieve high levels of control. The microgeographical breeding structure of the tsetse fly, G. pallidipes, was investigated by analysing spatial and temporal variation at eight microsatellite loci to test hypotheses about endemism and immigration. Samples were obtained at seasonal intervals from trap sites separated by 200 m to 14 km and arranged into blocks. G. pallidipes populations nearest to Lambwe and Nguruman also were sampled. Spatial analysis indicated that genetic differentiation by genetic drift was much less among trapping sites within Lambwe and Nguruman (FST ≤ 0.049) than between them (FST = 0.232). FST between Serengeti and Nguruman was 0.16 and FST between Kodera Forest and Lambwe was 0.15. The genetic variance in G. pallidipes explained by dry and wet seasons (0.33%) was about one-fifth the variance among collection dates (1.6%), thereby indicating reasonable temporal stability of genetic variation. Gene frequencies in Kodera and Serengeti differed greatly from Lambwe and Nguruman, thereby falsifying the hypothesis that Lambwe and Nguruman were repopulated by immigrants. Harmonic mean effective (= breeding) population sizes were 180 in Lambwe and 551 in Nguruman. The genetic data suggest that G. pallidipes in Lambwe and Nguruman have been endemic for long intervals.