Understanding the amount and distribution of genetic diversity in natural populations can inform the conservation strategy for the species in question. In this study, genetic variation at eight nuclear microsatellite loci was used to investigate genetic diversity and population structure of wild litchi (Litchi chinensis Sonn. subsp. chinensis). Totally 215 individuals were sampled, representing nine populations of wild litchi. All eight loci were polymorphic, with a total of 51 alleles. The expected heterozygosity in the nine populations ranged from 0.367 to 0.638 with an average value of 0.526. Inbreeding within wild litchi populations was indicated by a strong heterozygote defect. Significant bottleneck events were detected in the populations from Yunnan and Vietnam, which could be responsible for lower levels of genetic diversity in these populations. Measures of genetic differentiation (F ST = 0.269) indicated strong differentiation among wild litchi populations. Significant correlation was found between genetic differentiation and geographical distance (r = 0.655, P = 0.002), indicating a strong isolation by distance in these populations. Bayesian clustering suggested genetic separation among three regional groups, namely, the western group, the central group and the eastern group. Some conservation strategies for wild litchi populations were also proposed based on our results.