The Spanish Purebred Horse (Pura Raza Española, PRE) is a breed that holds high value in Spain with a notable economic impact. The genetic basis of skeletal muscle fiber type traits in the PRE has not been thoroughly studied. We genotyped 151 samples of PRE with data on muscle fiber. This data was derived from previous records of the Muscle Biopathology Laboratory of the University of Cordoba when biopsy samples were taken in 1998 from the right gluteus medius muscle of each horse at depths of two cm (superficial region), four cm (middle region), and six cm (deep region). We assessed sixteen phenotypic muscle fiber type variables and using a chip containing 71,607 markers. Statistical analyses identified of three single nucleotide polymorphisms (SNP) at the genome-wide level displaying highly significant associations that were all repeated in two muscle fiber type traits at 2 cm of depth: the proportion of the myosin heavy chain (MHC) isoforms IIA and IIX ( MHCIIA and MHCIIX). In proximity to these significant SNP, we discovered three genes— ABLIM2, SH3TC1 and HTRA3—providing valuable insights into the genetic factors influencing muscle fiber type traits in the PRE. This study represents a crucial first step towards identifying the genetic factors that influence muscle fiber type traits in the PRE.