Knowledge on population diversity and structure is of fundamental importance for conifer breeding programs. In this study, we concentrated on the development and application of high-density single nucleotide polymorphism (SNP) markers through a high-throughput sequencing technique termed as specific-locus amplified fragment sequencing (SLAF-seq) for the economically important conifer tree species, Chinese fir (Cunninghamia lanceolata). Based on the SLAF-seq, we successfully established a high-density SNP panel consisting of 108,753 genomic SNPs from Chinese fir. This SNP panel facilitated us in gaining insight into the genetic base of the Chinese fir advance breeding population with 221 genotypes for its genetic variation, relationship and diversity, and population structure status. Overall, the present population appears to have considerable genetic variability. Most (94.15%) of the variability was attributed to the genetic differentiation of genotypes, very limited (5.85%) variation occurred on the population (sub-origin set) level. Correspondingly, low F ST (0.0285-0.0990) values were seen for the sub-origin sets. When viewing the genetic structure of the population regardless of its sub-origin set feature, the present SNP data opened a new population picture where the advanced Chinese fir breeding population could be divided into four genetic sets, as evidenced by phylogenetic tree and population structure analysis results, albeit some difference in membership of the corresponding set (cluster vs. group). It also suggested that all the genetic sets were admixed clades revealing a complex relationship of the genotypes of this population. With a step wise pruning procedure, we captured a core collection (core 0.650) harboring 143 genotypes that maintains all the allele, diversity, and specific genetic structure of the whole population. This generalist core is valuable for the Chinese fir advanced breeding program and further genetic/genomic studies. breeder to address the population genetic architecture (genetic structure, diversity and relationship, etc.) and facilitate the accurate mating designs with parental analysis to accelerate the breeding progress [8]. The accessible molecular marker includes: (1) hybridization-based type such as restriction fragment length polymorphism (RFLP) and its modified forms, (2) PCR and electrophoresis-based type including random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), sequence-related amplified polymorphism (SRAP), inter simple sequence repeat (ISSR), and simple sequence repeat (SSR) etc., and (3) sequencing-based type dominated by single nucleotide polymorphism (SNP) [9][10][11]. Of these, SNPs have proved to be the most abundant form of variation within a species at genome level that provides more detailed insights into the genetic base of a population [10]. They have become more and more popular because they can be high-throughput detected through next-generation sequencing (NGS) at a moderate cost. Notably, the NGS-based SNP genoty...