Technical Abstract: A (GAAAAT)n repeat microsatellite was isolated from a partial Ostrinia nubilalis genomic library. Pedigree analysis indicated the marker was female specific, and referred to as Ostrinia nubilalis W-chromosome marker 1 (ONW1). Polymerase chain reaction (PCR) and DNA sequence analysis indicated that intact repeats of three, four, or five GAAAAT units were responsible for observed allele size differences. ONW1 amplification from 450 O. nubilalis females from ten North American locations indicated no significant ONW1 allele frequency differences were present between subpopulations or ecotypes. Low ONW1 mutation rate was inferred from population data, and attributed to reduced number of cell divisions during oogenesis, low repeat numbers, or short array length. Lack of meiotic crossover and fixation of nucleotides flanking the repeat may contribute to success of interspecies PCR amplification. The microsatellite was the first sex-linked microsatellite from a lepidopteran species, and had the unusual property of being highly conserved among related crop pest species. ONW1 may be informative for evaluation of ancient relationships and inferring the rate of lepidopteran sex chromosome evolution.