Objective: In order to provide a basis for advanced-cycle genetic improvement and sustainable utilization of Larix kaempferi, genetic diversity and its variation trend of the introduced provenance population(IP), the first cycle breeding population(FP) and the second cycle breeding population(SP) of L. kaempferi in northern subtropical subalpine region were compared and evaluated using EST-SSR markers. Method: 16 SSR markers were used to analyze the genetic diversity of 873 individuals from three populations of L. kaempferi. Estimation of genetic diversity parameters, analysis of molecular variance(AMOVA) and principal coordinate analysis(PCoA) were carried out using GenAlex 6.41. Result: A total of 106 alleles were detected at 16 pairs of primers in all the samples with an average of 6.7 alleles. The polymorphism of different loci was significantly different, among which 14 loci were moderately to highly polymorphic, indicating that these markers could well reflect the genetic diversity level of the population. The average effective allele number (Ne), Shannon diversity index (I) and polymorphism information content (PIC) of the three populations were 2.543, 0.979, and 0.480, respectively, indicating that all of the three populations had high level of genetic diversity. The diversity index I of IP, FP and SP were 0.911, 1.017 and 1.009, and the PIC were 0.432, 0.484 and 0.488, respectively. These indicated that the genetic diversity parameters of FP and SP were slightly higher than those of IP and there was no significant difference among the three groups. In general, the genetic distances among three populations were small, ranging from 0.006 to 0.075. The genetic distance between IP and FP was higher than that between IP and SP, while the genetic distance between FP and SP was the least. AMOVA analysis also showed that the differentiation among the three populations was small, and the variation mainly came from within the populations. Allele frequency comparison and PCoA analysis showed that the genetic basis of IP was obviously different from that of the other two populations. The genetic diversity level of SP could be effectively improved by introducing a small number of genotype with large differences from SP in IP. Conclusion: The genetic diversity of L. kaempferi breeding populations in northern subtropical region were high and the level of genetic diversity of improved population did not decrease compared with the introduced introduced provenance population. It was suggested that current breeding strategies in the region were appropriate for maintaining genetic diversity. The differences of origins among the three populations, pollen contamination in breeding process and high intensity of directional selection were the main causes for the differences in genetic composition and genetic diversity between the introduced provenance population and the first and the second cycle breeding populations. Replenishing the original resources into the breeding population in time is particularly important for the construction of advanced-cycle breeding populations of exotic species like L. kaempferi. © 2021, Editorial Department of Scientia Silvae Sinicae. All right reserved.