Polymorphisms of the HSPA2 gene; a member of the bovine HSP 70 genes family, have been associated with heat tolerance and reproductive performance in cattle. This study aims to determine the role of genetic variation in the HSPA2 gene in Holstein cattle fertility. A total of 366 cows were classified into anestrum (37.7%) and fertile (62.3%) groups. Reproductive and productive data were collected from farm records including age at first calving (AFC), calving interval (CI), calf birth weight, milk yield, and days of milking. The genomic DNA of the cows was isolated, and PCR-SSCP was conducted to detect three fragments of the HSPA2 gene; 208, 317, and 393 bp. HSPA2 F1 revealed three different patterns, HSPA2 F2 was monomorphic, and HSPA2 F3 showed only two patterns. Sequence analysis of HSPA2 F1 revealed three genotypes (CC, CT, and TT) with only one SNP (C/T) as a transition single base substitution mutation located at 10:76649474 with the ID rs377789074. Meanwhile, HSPA2 F3 showed two genotypes (CC and CT) with a synonymous SNP (C/T) located at 10:76680875 with the ID rs132895070. The CT genotype of HSPA2 F1 and HSPA2 F3 genes was associated with a shorter calving interval and heavier calf birth weight in both fertile and anestrum cattle. Our findings suggest that genetic variations in the HSPA2 gene could be utilized as a molecular marker for genetic selection to enhance reproductive performance in both fertile and anestrum Holstein cows raised in Egypt.