This paper reports the first direct molecular evidence that X chromosome loss during determination of male aphids (XO) is random. Clonal and sexual females, and males, of the species Sitobion near fragariae were screened using three polymorphic microsatellite loci. Two loci, Sm10 and Sm17, showed the same heterozygous genotypes in all three aphid morphs. The third, Sm11, was heterozygous for the same two alleles in clonal and sexual females, but of the 25 males screened 11 showed the ‘160’ allele and 14 showed the ‘156’ allele. This result indicates X-linkage of locus Sm11, with random loss of the X chromosome during the formation of male embryos. The possible implications of this result are discussed with respect to aphid sex determination, recombination and chromosome evolution.