<p>Research on the characteristics of the mineral profile of hooves can help prevent overgrowth or micro-cracks. Knowledge about the interaction of these elements shows how imbalances can lead to hoof pathology or suboptimal hoof quality. Sulphur plays a key role in keratin synthesis, contributing to hoof elasticity and strength. Iron is involved in oxygen transport to the hoof matrix cells, supporting proper metabolism and tissue regeneration. Zinc promotes keratinocyte differentiation and accelerates healing of micro-injuries within the horn structure. Copper is essential for the formation of disulfide bonds in keratin, ensuring the integrity and hardness of the hoof horn. Calcium affects cellular cohesion and mineralization of the horn matrix, stabilizing the horn structure at the microscopic level. The aim of the study was to assess the influence of gender and breed of horses on the content of Zn, Cu, Fe, Ca and S and their mutual correlations in hooves. Samples from 22 Polish sport horses (11 mares and 11 geldings) and 24 purebred Arabian horses (11 mares and 13 geldings) were tested. The collected tissue was washed and subjected to wet mineralization. Metal content was analysed using atomic absorption spectrometry, while S content was measured via the Bardsley-Lancaster method. Results showed significantly higher Ca and Zn levels in Arabian horses, while Polish sport horses had higher Cu content. Significant gender differences were observed only for S in Polish sport horses. Correlation analysis revealed significant negative correlations between Ca and Zn in Polish sport horses (<i>p</i> = 0.0208) and Ca and Fe in Arabians (<i>p</i> = 0.0118).</p>