Analyzing genetic variability and inbreeding trends is essential for effective breed management in animal populations. To this, the characterization of runs of homozygosity (ROH) provides a good genomic approach to study the phenomena. The Polo Argentino (PA) breed, globally recognized as the best adapted to playing polo, is known for its strong influence of Thoroughbreds, intense selective breeding, and extensive use of reproductive biotechnologies. This study investigates the PA’s genomic variability, by characterizing the ROH landscape and identifying ROH islands (ROHi) as potential genomic footprints for the breed. PA horses (n = 506) were genotyped using EquineGGP™ array v5 (70 k). We calculated the inbreeding coefficient based on ROH (FROH—ancestral and recent) using a chromosomal approach. Finally, we identified genomic regions with increased ROH frequency (ROHi) and their associated genes. An average of 79.5 ROH per horse was detected, with a mean length of 4.6 Mb. The average FROH was 0.151, but most of them (54%) corresponded to ancestral inbreeding (ROH < 5.5 Mb). However, 4 ROHi were identified in ECA 1, 3, 7 and 17, containing 67 genes, some of which were related to behavior, neurodevelopment, and metabolic functions. This genomic analysis determined, for the first time, the length and location of homozygosity segments in the PA breed and identified ROHi associated with potential genomic regions and genes for positive selection in the breed.