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The phenomenon of chimaerism occurs in the majority of cattle twin pregnancies. The objectives of this study were to develop a powerful diagnostic test for chimaerism in bovine male and female co-twins using X and Y chromosome-linked markers and to determine the extent of chimaerism in twins, triplets and quadruplets. We developed a multiplex PCR set of three polymorphic markers on chromosome X (DIK2865, DIK2283, AGLA257), where the presence of >1 and >2 alleles per marker is sufficient to prove chimaerism in males and females, respectively. In addition, a specific segment on chromosome Y (BOV97M) is included in the set to indicate chimaerism in females. Visualization of chimaeric alleles was best for DNA extracted from blood, fair for DNA from vaginal smears and failed for DNA extracted from hair. The power of chimaerism identification using this set of markers for DNA extracted from blood was calculated as 99% in males and virtually 100% in females. All females and males in heterosexual twins, triplets and quadruplets displayed evidence of a chimaeric allele in at least one and maximum of three of three X chromosome markers analysed. In addition, all females showed the presence of the BOV97M segment and were validated as chimaeric by the standard clinical diagnosis of impaired vaginal length. Quantitative PCR analysis of BOV97M copies in all twins vs. their sires showed a mean ratio of 45–68% in females and 39–49% in males, indicating a substantial symmetrical exchange of cells among all co-twins. The proposed analysis of X and Y chromosome-linked markers is advantageous to previous methods based on Y chromosome sequences only, because it detects chimaerism in both male and female co-twins.