PCR amplification of the six fragments of regulatory and coding regions of chicken ChEST985k21 gene (accession no. CR523443), substantially affecting the egg shell thickness quantitative trait, was carried out. Sequencing of these fragments in six chickens from a native Polish breed, Green-legged Partridgenous, with different manifestation of the trait of interest enabled identification of six single nucleotide polymorphism (SNP) sites within the ChEST985k21 sequence. Five of these sites were located in the regulatory region, and one site, in the coding region. For all SNPs identified, the existence of transcription factor binding sites, present in only one allelic variant, was demonstrated. This finding enables considering these sites as regulatory single nucleotide polymorphisms, rSNP. The effect of rSNP discovered on the chicken egg shell thickness was tested using PCR amplification with allele-specific primers. In the groups of chicken of Rhode Island Red breed with thick (389.9 +/- 13.09 microm) and thin (315.7 +/- 21.38 microm) egg shells statistically significant differences in the allele frequencies of the ST2_1, ST3_1, ST3_2, and ST3_3 polymorphic loci. In the same groups of birds, statistically significant differences in the shell thickness were observed in the rSNP allele genotypic classes ST2_1, ST3_1, ST3-2, ST3_3, and ST6_1. Based on these data, it was concluded that rSNPs influenced manifestation of the quantitative trait examined, and the genotyping system for marker assisted selection was constructed.