The economic efficiency of sheep breeding, aiming to enhance productivity, is a focal point for improvement of sheep breeding. Recent studies highlight the involvement of the Early Region 2 Binding Factor transcription factor 8 ( E2F8) gene in female reproduction. Our group's recent genome-wide association study (GWAS) emphasizes the potential impact of the E2F8 gene on prolificacy traits in Australian White sheep (AUW). Herein, the purpose of this study was to assess the correlation of the E2F8 gene with litter size in AUW sheep breed. This work encompassed 659 AUW sheep, subject to genotyping through PCR-based genotyping technology. Furthermore, the results of PCR-based genotyping showed significant associations between the P1-del-32bp bp InDel and the fourth and fifth parities litter size in AUW sheep; the litter size of those with genotype ID were superior compared to those with DD and II genotypes. Thus, these results indicate that the P1-del-32bp InDel within the E2F8 gene can be useful in marker-assisted selection (MAS) in sheep.