Landraces of seed and dessert type watermelons from three provinces of Mozambique with distinct agro-ecological conditions, including humid and semi-dry areas from the north to the south of the country, were analysed to assess their genetic differentiation. Ninety-six accessions (269 plants) were tested with 24 microsatellite markers resulting in 110 alleles. Analysis of molecular variance showed that 63% of the total variation in the plant material could be explained among the accessions, while 37% of the variation was within accessions. Molecular variance between material used for seed extraction and dessert consumption explained 34% of the total variation whereas villages explained 27% of the molecular variation among the sampled locations. Structure analysis revealed that the material could be differentiated into three genetic groups. The seed types clustered in one genetic group, irrespective of provincial origin. Dessert type accessions from the semi-arid south and the central part were assigned to a second genetic group, while accessions from the northern more humid region of the country were assigned to a third genetic group. The observed genetic diversity may reflect farmer selection under different agro-ecological conditions or an introduction of material from different sources into the growing areas.