Aggressive behavior is a major welfare and production challenge in cage-free laying hen systems, but the genetic mechanisms behind trait variation are poorly understood. This study investigated the role of a previously identified aggression-associated SORCS2 polymorphism and evaluated the expression of SORCS2, DRD1–4, and NGF in laying hens. A total of 360 hens from three experimental populations were subjected to behavioral observations. The SORCS2 SNP was genotyped by PCR-RFLP, pituitary gene expression was measured by qPCR, and specific-locus amplified fragment sequencing (SLAF-seq) was performed on pooled DNA samples representing the least (LA) and the most (MA) aggressive hens. The previously reported SORCS2 polymorphism was detected in all populations but showed no significant (p >0.05) association with behavioral traits. Among the analyzed genes, SORCS2 expression differed (p < 0.05) between populations and was consistently higher in the MA compared to the LA hens, whereas DRD1–4 and NGF expressions were similar (p > 0.05) among populations and aggression-divergent groups. The exploratory gene-level SNP differentiation analysis placed SORCS2 in the top 1.3% of genes between aggression-divergent groups, indicating a higher relative ranking than in population-based comparisons (top 4.5%). Overall, an exploratory association between SORCS2 and aggressive phenotype was detected in the experimental populations, which supports further investigation of this gene in relation to chicken behavior.