The barley male sterility gene (msg6) located on chromosome 6H has been used in breeding and research since its discovery 7 decades ago, but to date, no research has been reported that linked the gene with molecular markers. The main objective of this study was to identify expressed sequence tag'simple sequence repeat (EST-SSR) markers linked to msg6 as this could provide opportunities for gene discovery. In a cross of a male sterile line (04-042B) with a fully fertile line (VB0330;VB9524/Mundah), male sterility segregated in a 3:1 ratio of fertile to completely sterile plants (v2 5 0.03, P0.05 5 0.95), in a population of 250 F2 plants. Multipoint linkage mapping placed the msg6 gene at 4.9 cM from the EST-SSR, GBM1267, whereas 2-point analysis estimated a recombination fraction of 0.05 ± 0.02 (logarithm of the odds score 5 26.34) between the EST-SSR and the male sterility gene. Multiple interval quantitative trait locus (QTL) analysis of spike weight, anindicative measure of reproductive success, identified a QTL near GBM1267 as having a major influence, explaining 68.7% of the variation in weight of individual spikes. The GBM1267 marker segregated in a 1:2:1 ratio, which makes it highly desirable for marker-assisted selection, as it can distinguish the recessive from the dominant and from heterozygous individuals. Another EST-SSR marker, designated VBMS103, was developed in the present study to provide an additional marker with known sequence (AL501881) close to the msg6 gene. The results provide highly informative functional tools for tracking the msg6 gene in breeding programs.