Methods <p>Bioinformatics was used to analyze the STR information of human chromosome 21, focusing on the 21q21.1-22.3 region to screen STR loci with 4–6 base repeat core sequences. 20 candidate STR loci were selected and identified by polymerase chain reaction (PCR) amplification, polyacrylamide gel electrophoresis separation and silver staining profiling technology. The STR loci with effective amplification and genetic diversity obtained by screening were further subjected to genetic survey and analysis in 500 unrelated Han individuals from Guizhou Province.</p> Results <p>Bioinformatics analysis showed that a total of 387,437 STR loci were detected on human chromosome 21, 90% of which were located in the long arm region; PCR amplification results of 30 individuals showed that 3 pairs of primers among the 20 candidate STR loci had excellent amplification effect, with clear bands and polymorphism; these 3 STR loci (named D21S020800, D21S163280 and D21S001752) were applied to the genetic survey of 500 Han individuals in Guizhou Province. The results showed that the 3 loci contained 39, 16 and 12 alleles respectively, and their genotype distributions all conformed to the Hardy-Weinberg law (P&gt;0.05); heterozygosity values were 0.8174, 0.6547 and 0.6464 respectively, the polymorphic information content (PIC) values were 0.9553, 0.8779 and 0.8053 respectively, the individual identification rates were 0.9936, 0.9644 and 0.9451 respectively, and the paternity exclusion probabilities were 0.6317, 0.3617 and 0.3503 respectively.</p> Conclusion <p>The newly discovered 3 STR loci on chromosome 21 (with 5-base repeat core sequences) have high heterozygosity and discriminative power, have good application prospects in the fields of individual identification, paternity testing, population genetic analysis and screening of hereditary diseases on chromosome 21.</p>