It is vital to measure the levels of genetic diversity and differentiation between populations in a species to understand the current genetic structure and evolution of the species. Here, MIG-seq (multiplexed inter-simple sequence repeat genotyping by sequencing) was employed to assess the genetic variation in two tropical leguminous tree species, Dalbergia cochinchinensis and D. nigrescens, in Cambodia and Thailand. Sequence data for 255–618 loci, each with an approximate length of 100 bp, were obtained, and the nucleotide diversity, Tajima's D and FST were computed. The estimates calculated from the data obtained by MIG-seq were compared to those obtained by Sanger sequencing of nine nuclear coding genes in D. cochinchinensis in our previous study. The nucleotide diversity at the MIG-seq loci was slightly higher than that at silent sites in the coding loci, whereas the FST values at the MIG-seq loci were generally lower than those at the coding loci, although the differences were not significant. Moreover, nucleotide diversities within populations of the two species were similar to each other, at approximately 0.005. Three and four population clusters were genetically recognized in D. cochinchinensis and D. nigrescens, respectively. Although the populations were differentiated from each other, the levels of differentiation among them, as measured by FST, were higher in D. cochinchinensis than in D. nigrescens. This indicates higher levels of gene flow between the populations in the latter species. We recommend using MIG-seq for quick surveys of genetic variation because it is cost-effective and results in smaller variance in the estimates of population genetic parameters.