MicroRNAs are an abundant class of small non-coding RNAs that regulate gene expression. Genetic variations in microRNA sequences may be associated with phenotype differences by influencing the expression of microRNAs and/or their targets. This study identified two single nucleotide polymorphisms (SNPs) in the genomic region of the microRNA miR-1596 locus of chicken. Of the two SNPs, one was 95 bp upstream of miR-1596 (g.5678784A>T) and the other was in the middle of the sequence producing the mature microRNA gga-miR-1596-3p (g.5678944A>G). Genotypic distribution of the two SNPs had large differences among 12 chicken breeds (lines), especially between the fast-growing commercial lines and the slow-growing Chinese indigenous breeds for the g.5678784A>T SNP. Only the g.5678784A>T SNP was significantly associated with residual feed intake (RFI) in the F2 population derived from a fast-growing and a slow-growing broiler as well as in the pure Huiyang bearded chicken. The birds with the AA genotype of the g.5678784A>T SNP had lower RFI and higher expression of the mature gga-miR-1596-3p microRNA of miR-1596 than did those with the other genotypes of the same SNP. We also found that the expression of the mature gga-miR-1596-3p microRNA of miR-1596 was significantly associated with RFI. These findings suggest that miR-1596 can become a candidate gene related to RFI, and its genetic variation may contribute to changes in RFI by altering expression levels of the mature gga-miR-1596-3p microRNA in chicken.