Complex chromosome rearrangements (CCRs) are structural aberrations involving three or more breakpoints on two or more chromosomes. These CCRs result in a high rate of chromosome imbalances potentially leading to subfertility and congenital abnormality. In this study, we analysed meiotic segregation in the sperm of a patient with a familial CCR 46, XY,t(1;19;13)(p31;q13.2;q31)mat included in an intracytoplasmic sperm injection program because of oligoasthenozoospermia. The rearrangement was first identified using conventional and molecular cytogenetic methods. Primed in situ labelling (PRINS) and fluorescence in situ hybridization (FISH) techniques were then combined allowing the simultaneous use of five fluorochromes on the same sperm preparation, for the segregation analysis and the evaluation of the reproductive options for this patient. Segregation analysis was performed in a total of 1822 sperm nuclei from the translocation carrier. The percentage of unbalanced sperm was 75.9%, including 34.1% from 3:3 segregation, 38.2% from 4:2 segregation, 3.5% from 5:1 segregation and 0.05% from 6:0 segregation. Only 14.8% of sperm nuclei were consistent with a normal or balanced chromosome complement. In conclusion, chromosome segregation analysis combining FISH and PRINS was performed in sperm from a CCR carrier using five fluorochromes. These results advance our understanding of the mechanisms of meiotic segregation, and facilitate the assessment of the usefulness of preimplantation genetic diagnosis procedures in CCR couples.