Purpose To analyse the segregation of a Robertsonian translocation t(13;14) in five male carriers, and to verify a possible inter-chromosomal effect (ICE) of the Robertsonian translocation on chromosomes 18, X, and Y. Methods The spermatozoa of these patients (n=5) and of 15 donors with normal semen parameters and 46,XY karyotype were analysed using triple colour FISH with locus specific probes for chromosomes 13, 14, and 21 and by triple colour FISH for chromosomes X, Y, and 18. Results The frequency of balanced spermatozoa resulting from alternate segregation varied between 62.16% and 81.70% with a mean of 71.5%. The rates of unbalanced spermatozoa resulting from adjacent segregation varied between 13.4% and 25.1% with a mean of 18.26%. Triple colour FISH X-Y-18 showed a significant increase in disomy frequencies of these chromosomes in comparison with controls, indicating an ICE. Conclusion In spite of the high number of normal/balanced frequencies, there remain many unbalanced spermatozoa resulting from adjacent mode of segregation. This raises the question of the unbalanced chromosomal risk for the offspring of 45,XY, t(13;14) males and the importance of the genetic counselling prior to ICSI or IVF treatment for couples where the male is a Robertsonian translocation carrier.