Background: Slash pine (Pinus elliottii) is an important timber species in southern China, having been introduced from North America over a century ago. Recently, dozens of improved slash pine varieties have been authorized. However, the absence of efficient molecular markers hinders the identification of these improved varieties. In this study, we aimed to construct a set of DNA fingerprinting based on SNP markers for improved slash pine varieties and provide a technical procedure for their identification. Results: The genotypes of 29 improved slash pine varieties were captured using 51K liquid-phase probes developed by our team, resulting in the genotyping of 560,567 SNPs through next-generation sequencing. A total of 3502 SNPs were retained after screening for minor allele frequency, missing rate, heterozygosity rate, and other parameters. Subsequently, a random forest model was used to reduce the number of SNPs to 50. The identification rate of improved varieties with these 50 SNPs achieved 98.64%. We concluded that these SNPs effectively distinguish the improved varieties, which enabled the construction of DNA fingerprints for the improved slash pine varieties. Conclusions: This study provides a technical procedure for identifying improved slash pine varieties based on the Random Forest model and constructs a DNA fingerprinting of the 29 slash pine varieties using 50 SNPs.