Lactarius hatsudake is an ecologically and economically significant wild edible mushroom in southern China. To elucidate its population genetic diversity, differentiation, and evolutionary history, we analyzed 172 fruiting bodies from eight geographic populations (AQ, BS, DZ, JS, NC, PT, SG, YX) across seven provinces in the western and eastern regions of southern China using five highly polymorphic simple sequence repeat (SSR) markers. Combined with STRUCTURE clustering, discriminant analysis of principal components (DAPC), unweighted pair group method with arithmetic mean (UPGMA), and analysis of molecular variance (AMOVA), the results revealed high polymorphism across the studied loci (mean PIC = 0.842). A total of 75 alleles were identified, averaging 15 alleles per locus. At the population level, the mean effective number of alleles (Ne) was 4.023, and the mean unbiased gene diversity (uH) was 0.768. The NC population exhibited the highest genetic diversity (uH = 0.796), whereas the BS population showed relatively lower diversity (uH = 0.647). Clustering analyses (STRUCTURE, DAPC, and UPGMA) consistently identified two distinct genetic clusters (K = 2). Cluster I consisted of populations AQ, PT, BS, and SG, while Cluster II was composed of the remaining four populations. Notably, individuals from AQ and NC displayed significant genetic admixture, suggesting a transitional zone. AMOVA revealed that the majority of genetic variation (83%) resided within populations and 17% among populations. Moderate population differentiation (ENA-corrected global Fst = 0.102) and admixture signals suggest non-negligible connectivity among populations.

Lactarius vividus is a wild edible ectomycorrhizal mushroom widely distributed in southern China, where it forms an obligate symbiotic relationship with Pinus species. However, its genetic diversity and population differentiation remain poorly understood. We collected 95 basidiomata from ten geographic populations spanning seven provinces across an east–west transect of southern China and developed seven SSR primer pairs at the genomic level to investigate its genetic diversity, population differentiation and spatial genetic structure. Genetic diversity showed a clear east-to-west declining trend, being highest in population AQ (Anhui Province) and lowest in KM (Yunnan Province). Observed heterozygosity (Ho) was lower than expected heterozygosity (He) in most population-locus combinations, and many tests showed significant departures from Hardy–Weinberg equilibrium (HWE), indicating a pronounced heterozygote deficit. Analysis of molecular variance (AMOVA) showed substantial population differentiation, with 63% of the total genetic variation occurring among populations. A Mantel test detected a significant positive association between genetic and geographic distances (r = 0.322, p = 0.032), consistent with isolation by distance (IBD). STRUCTURE identified three genetic clusters, while DAPC and UPGMA analyses further revealed geographic differentiation and admixture or genetic overlap among some samples. These findings provide a basis for prioritising geographically distinct populations in the conservation and sustainable management of this wild edible mushroom.