Offspring of a highly inbred gynogenetic line of Oreochromis aureus displayed 12-fold increase in twinning rate compared to the outbred population. Asymmetric conjoined twins, which consist of a normal embryo attached to a malformed-atrophic twin, were frequently encountered in both gynogenetic (90·7%) and outbred (38·2%) embryos. The monozygotic origin of these twins was determined using five microsatellite markers. Progeny of heterozygous parents for the microsatellite UNH159 were separated into sub-sets of twins and normal full-sibs. Consistent with previous reports, the normal embryo sub-set exhibited elimination of both types of homozygotes for the UNH159 genetic marker at 2–8 days after fertilization. Unexpectedly, this elimination was less frequent in twins. The UNH159 marker as well as RNA-binding motif protein, X-linked (rbmx), SRY-box containing gene 3 (sox3) and alpha-thalassemia/mental retardation syndrome X-linked (atrx) genes were mapped to linkage group 2. These gene orthologues are all located on the mammalian X chromosome and atrx is necessary for the X-chromosome inactivation.