To determine the segregation pattern of the translocated chromosomes in spermatozoa of human males with translocations.Retrospective case–control study.Hospital-based genetic laboratory for reproductive biology.A carrier with Y–autosome reciprocal translocation, two with autosome–autosome reciprocal translocations, and one with Robertsonian translocation.Blood sample and sperm sample collection from each translocation carrier.Fluorescence in situ hybridization on lymphocyte slides to characterize each translocation case. Fluorescence in situ hybridization with specific DNA probes for each of the sperm samples to characterize the chromosomes involved in the rearrangement and to evaluate the possible interchromosomal effect for chromosomes 18, X, and Y.Each translocation carrier showed a specific mode of segregation pattern of the translocated chromosomes, confirming the dependence on chromosomes involved in the translocation. The highest frequency from alternate segregation was with the carrier of Robertsonian translocation (90.9%), and the lowest was with the carrier of Y–autosome translocation (29.7%). No evidence of an interchromosomal effect for chromosomes 18, X, and Y were detected.Depending on the rate of the genetically normal and abnormal segregation modes, we can evaluate the chance of having a healthy proband. These results ensure more accurate genetic counseling for patients in assisted reproduction centers.