To identify all sesame chromosomes, a multicolour fluorescence in situ hybridization procedure was developed to delineate and physically map the probes of simple sequence repeat (AC)8, 45S rDNA, 5S rDNA, cot-1 DNA and telomeric repeat (TTTAGGG)n. The results showed that the karyotype formula of this species was found to be 2n=6m+18sm+2st. All 13 chromosomes were identified by the banding and colour pattern in the three examined varieties. The FISH analysis of rDNA, (AC)8 and Cot-1 DNA sites showed two sites for 5S rDNA, three sites for 45S rDNA, nine sites for (AC)8 and ten sites for cot-1 DNA. The (AC)8 on the long arm, pericentromeric region of short arm, at the centromeric and near telomeres at the short arm was assigned number 1, 2, 8 and 12, respectively, by simultaneous hybrization with a Cot-1 DNA site. On chromosome 3, we found two (AC)8 sites. One chromosome with no other hybridization signals but a (AC)8 site on the short arm near telomeres was assigned number 11. Both chromosome numbers 5 and 7 had the 45S rDNA and Cot-1 DNA sites near telomeres at the short arm, while the chromosome number 5 was longer than the 7th. The chromosome numbers 6 and 9 had the (AC)8, 5S rDNA and Cot-1 DNA sites near telomeres at the short arm, while the chromosome number 6 was longer than the 9th. The chromosome with the 45S rDNA site at the telomeres and Cot-1 DNA site near the telomeres at the short arm was identified as number 10. The chromosomes with no other hybridization signals but telomeres sites were identified 4 and 13, while the chromosome number 4 was longer than the 13th. It is hoped that the results in this study would provide the reference and support for further studies in genetics and evolution in sesame.