The aim of this study was to determine new polymorphisms in GDF5 (growth differentiation factor 5 (CDMP-1)) and EPS8 (epidermal growth factor receptor substrate 8). After the PCR analysis of the identified gene regions of 72 head Holstein cattle, samples were separated by the high-resolution melting analysis (HRMA), and sequence analysis was applied to some randomly selected samples. New polymorphisms were identified in the determined genes and gene regions as a result of the sequence analysis. In the 1st exon of the EPS8 gene, 9 polymorphic regions were identified as g.94554132C>T, g.94554252G>C, g.94554348T>G, g.94554354C>G, g.94554372C>G, g.94554389A>G, 94554392C>G, 94554399A>G, and 94554439C>G. Within the region examined in the 2nd exon of the EPS8 gene, only the g.94555920T>G polymorphism was identified. In the 1st exon of the GDF5 gene, the g.65340723G>A and g.65340727T>C polymorphisms were identified. In the 2nd exon of the GDF5 gene, the g.65340902A>G polymorphism was identified. No polymorphism was found in the examined 2nd intron region of the GDF5 gene. As a result, new polymorphic regions were detected on EPS8 and GDF5 genes by HRMA, genotype and allele gene frequencies of polymorphic regions were determined. Hardy-Weinberg equilibrium test generally shown that the distribution of genotype frequencies in polymorphic regions is in genetic equilibrium.