Trait variation among natural populations and their cultivated relatives occurs due to evolutionary forces, including selection and drift. In the present study, we analyzed these forces at the locus level in a global barley diversity set using population genetics analysis. Genome-wide outlier loci detection found a locus on chromosome 2H at which a common single nucleotide polymorphism (SNP) marker SCRI_RS_170235 accounted for the highest diversity index (Fst) values between cultivars and landraces and between cultivars and wild accessions. For a population wide genetic analysis, we developed a Polymerase Chain Reaction (PCR)-based cleaved amplified polymorphic marker at the identified locus. Marker genotyping of 115 genotypes identified a characteristic distribution of polymorphisms among the cultivated, landraces, and wild barley accessions. Using this marker, we screened a library of wild barley introgression lines (IL) and selected IL S42IL-109 that carried the wild introgression of the outlier locus in cultivar ‘Scarlett’ background. A plethora of phenotypic evaluation was performed between the S42IL109 and ‘Scarlett’ to dissect the putative effect of the identified outlier locus. Comparison of S42IL109 and ‘Scarlett’ revealed significant difference in the development of phyllochron two (Phyl-2), phyllochron three (Phyl-3), and phyllochron four (Phyl-4). Across the three phyllochrons, it was consistently observed that S42IL109 developed successive leaves in a shorter time span, by one to two days, compared to ‘Scarlett’. These data suggest that outlier locus may influence phyllochron variation which underwent positive selection in barley.