Natural stands of sawara cypress (Chamaecyparis pisifera), an important conifer species endemic to Japan, are fragmented and declining. This raises concerns about the loss of genetic diversity and potential genetic swamping via frequent gene flow from widespread surrounding plantations of the closely related hinoki cypress (Chamaecyparis obtusa). To determine the extent of interspecific hybridization between the two species, we conducted genetic analyses of mature trees and seedlings growing in the natural forests. Using five nuclear microsatellite (SSR) markers, we examined the genetic diversity of hinoki cypress (N = 123) and sawara cypress (N = 80) in the Kuraiyama Experimental Forest of Gifu University. The genetic differentiation coefficient (G'_ST) of 0.442 and the results of Bayesian cluster analysis revealed clear differentiation between the two species. Analysis of 175 Chamaecyparis seedlings collected from the forest floor of the same natural forest using same SSRs and cleaved amplified polymorphic sequences markers revealed their species identities, with none of the seedlings identified as interspecific hybrids. Given the clear genetic differentiation between the natural forest populations of the two species and the absence of hybrids among the seedlings, we conclude that gene flow between the two species under natural conditions is highly restricted or extremely rare.