Longan (Dimocarpus longan Lour.) is grown commercially in many countries, including China, Thailand, the Philippines, Malaysia, Vietnam, India, Australia, and Hawaii. Thailand is the second largest producer and largest exporter of longan in the world. Currently, there is limited information on the genetic background, population structure, and genetic relationships among longan cultivars in Thailand. In this study, a total of 50 longan accessions from a community-based germplasm collection in Thailand were analyzed using 10,619 SNPs from genotyping-by-sequencing (GBS). Based on the results of STRUCTURE analysis, 43 accessions were classified into 4 subpopulations, and the other 7 accessions were found to contain admixed genotypes. Based on UPGMA clustering analysis and PCoA analysis, the longan accessions could be divided into six major groups consistent with those identified by STRUCTURE. A relatively high degree of genetic variation was observed among the longan accessions, as quantified by the expected heterozygosity (He = 0.308). AMOVA results showed that 74% and 26% of the total variation occurred between and within populations, respectively. Obvious genetic differentiation between populations (FST = 0.25) was observed. The results of this study are useful for managing longan germplasm and may facilitate the genetic improvement of longan.