<p>The thousand kernel weight trait (TKW) has been selected by oat (<i>Avena sativa</i> L.) breeders worldwide to increase both grain yield and grain quality. However, genomic regions controlling TKW on oats need to be better understood, especially on oats adapted to subtropical environments. This study aimed to identify genomic regions controlling TKW in oats through a genome-wide association study (GWAS). To elucidate the genetic architecture of TKW in subtropical conditions, a panel of 383 oat genotypes, primarily adapted to the subtropical zone, was evaluated in three environments. The genomic analysis investigated the association among 11,559 single nucleotide polymorphisms (SNPs) molecular markers and TKW. Fifteen markers associated with TKW were identified on chromosomes 3A, 2C, and 5C. Markers located on chromosomes 3A and 2C exhibited strong and stable statistical significance across environments for TKW. Furthermore, when comparing the significant marker alleles of old and modern varieties of the panel, it was found that the old varieties tended to possess inferior alleles, whereas the more modern varieties, with higher TKW, tended to carry superior alleles, with exceptions. Analyzing genes closely located to the highly significant markers on chromosomes 3A and 2C, different gene functions, ranging from stress tolerance to cellular process, were observed. These results provided promising information about the genomic regions controlling TKW in subtropical environments, which could assist oat breeding programs in implementing molecular breeding strategies to select superior genotypes for TKW on oats.</p>