Mango (Mangifera indica L.) is an important fruit crop with a long cultivation history in Myanmar. This study evaluated the genetic variation within two economically important traditional varieties, ‘Yin Kwe’ and ‘Sein Ta Lone’, and the relationship between genetic variation and propagation practices. Genetic variation was estimated by genotyping 94 individuals with 12 single sequence repeat markers. ‘Yin Kwe’ (n = 53) showed higher levels of observed heterozygosity (Ho = 0.59) and average genetic distance among individuals (Da = 0.29) than did ‘Sein Ta Lone’ (n = 41; Ho = 0.45; Da = 0.09). The differences between the two varieties at the DNA level were significant (Fst = 0.44). The broader genetic background in ‘Yin Kwe’ compared with ‘Sein Ta Lone’ was also demonstrated by neighbour-joining and principal coordinates analyses. Differences in variety uses and propagation practices were determined by interviewing local specialists in Lower Myanmar (southern Myanmar). ‘Yin Kwe’ was often used as a rootstock for ‘Sein Ta Lone’. Clonal propagation by grafting was observed frequently for ‘Sein Ta Lone’ but never for ‘Yin Kwe’. The differences in genetic variation between these two varieties might have been caused by the propagation practices for each variety, which result from their respective uses.