To provide an efficient tool for the identification of individuals and paternity testing in the breeding of Pinus thunbergii Parl., a battery of 18 single nucleotide polymorphism (SNP) markers was developed. Multiplexed single nucleotide primer extension was utilized as the assay system, which facilitated automated high-throughput analysis. The expected heterozygosity and polymorphic information content were 0.342–0.500 (average 0.434), and 0.283–0.375 (average 0.339), respectively. The cumulative confusion probability and discrimination power were 3.40 × 10−8 and 1 − 3.40 × 10−8, respectively, whereas the probabilities of paternity exclusion when only one parent was genotyped and when the genotypes of both suspected parents were known were 0.965 and 0.996, respectively. The assay system was applied to investigate the mating dynamics of four clonal seed orchards. By genotyping the 18 nuclear SNPs in both the biparentally derived diploid embryo and the corresponding maternally derived haploid megagametophyte, the maternity and paternity of 1,915 seeds could be assigned accurately. The paternity analysis indicated that, for 64.5 % of the seeds, both parents were found in the orchard. The analysis of relative maternal contribution revealed that, in all the seed orchards, one clone of the 15 in the orchards did not act as a mother. The analysis of relative paternal contribution showed that all clones functioned as fathers; however, the degree of contribution differed considerably among the clones. The SNP markers, in combination with separate analysis of two seed tissues (embryo and megagametophyte), were an effective means of investigating mating dynamics in P. thunbergii.