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Species delimitation has been a major research focus in evolutionary biology. However, the genetic delimitation of recently diverged species varies depending on the markers examined. In this study, we aimed to examine genetic differentiation and delimitations between only two species of Pugionium Gaertn (Brassicaceae)-Pugionium cornutum (L.) Gaertn and Pugionium dolabratum Maxim-that occur in the desert habitats of central Asia and have parapatric distributions. We genotyped 169 individuals from 25 populations, using two chloroplast (cp) DNA fragments (trnV-trnM and trnS-trnfM), seven simple repeated sequence (SSR) loci and the nuclear ribosomal internal transcribed spacer (ITS). Four cp haplotypes were identified, three of which commonly occur in the two species, suggesting incomplete species-specific lineage sorting. Between-species cpDNA differentiation (F (CT)) was low, even lower than among populations of the same species. However, we found higher than average SSR F (CT) values, and both Bayesian clustering of SSR variables and maximum-likelihood genetic analyses divided all sampled individuals into two groups, agreeing well with morphological separation, although gene flow between species was obvious according to the SSR loci data. However, two types of ITS sequences were highly consistent with the morphological delimitation of the two species in all sampled individuals. These results together suggest that these two species shared numerous ancestral cpDNA polymorphisms and point to the importance of nuclear DNA (ITS or genetic accumulation at multiple loci) in delimiting recently diverged species.