ABSTRACT.- Temperament can be defined as the emotional variability among animals of the same species in response to the same stimulus, grouping animals by their reactivity as nervous, intermediate, or calm. Our goal was to identify genomic regions with the temperament phenotype measured by the Isolation Box Test (IBT) by single-step genome-wide association studies (ssGWAS). The database consisted of 4317 animals with temperament records, and 1697 genotyped animals with 38,268 effective Single Nucleotide Polymorphism (SNP) after quality control. We identified three genomic regions that explained the greatest percentage of the genetic variance, resulting in 25 SNP associated with candidate genes on chromosomes 6, 10, and 21. A total of nine candidate genes are reported for the temperament trait, which is:PYGM, SYVN1, CAPN1, FADS1, SYT7, GRID2, GPRIN3, EEF1A1 and FRY, linked to the energetic activity of the organism, synaptic transmission, meat tenderness, and calcium associated activities. This is the first study to identify these genetic variants associated with temperament in sheep, which could be used as molecular markers in future behavioral research. © 2024 by the authors.