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Background: Fragmentation of tsetse habitat in eastern Zambia is largely due to encroachments by subsistence farmers into new areas in search of new agricultural land. The impact of habitat fragmentation on tsetse populations is not clearly understood. This study was aimed at establishing the impact of habitat fragmentation on physiological and demographic parameters of tsetse flies in order to enhance the understanding of the relationship between fragmentation and African animal trypanosomosis (AAT) risk., Methods: A longitudinal study was conducted to establish the age structure, abundance, proportion of females and trypanosome infection rate of Glossina morsitans morsitans Westwood (Diptera: Glossinidae) in areas of varying degrees of habitat fragmentation in Eastern Zambia. Black screen fly rounds were used to sample tsetse populations monthly for 1 year. Logistic regression was used to analyse age, proportion of females and infection rate data., Results: Flies got significantly older as fragmentation increased (p < 0.004). The proportion of old flies, i.e. above ovarian category four, increased significantly (P < 0.001) from 25.9% (CI 21.4-31.1) at the least fragmented site (Lusandwa) to 74.2% (CI 56.8-86.3) at the highly fragmented site (Chisulo). In the most fragmented area (Kasamanda), tsetse flies had almost disappeared. In the highly fragmented area a significantly higher trypanosome infection rate in tsetse (P < 0.001) than in areas with lower fragmentation was observed. Consequently a comparatively high trypanosomosis incidence rate in livestock was observed there despite lower tsetse density (p < 0.001). The overall proportion of captured female flies increased significantly (P < 0.005) as fragmentation reduced. The proportion increased from 0.135 (CI 0.10-0.18) to 0.285 (CI 0.26-0.31) at the highly and least fragmented sites, respectively., Conclusions: Habitat fragmentation creates conditions to which tsetse populations respond physiologically and demographically thereby affecting tsetse-trypanosome interactions and hence influencing trypanosomosis risk. Temperature rise due to fragmentation coupled with dominance of old flies in populations increases infection rate in tsetse and hence creates high risk of trypanosomosis in fragmented areas. Possibilities of how correlations between biological characteristics of populations and the degree of fragmentation can be used to structure populations based on their well-being, using integrated GIS and remote sensing techniques are discussed.