Three varieties of Lathyrus sativus L. released from Bangladesh Agricultural Research Institute (BARI), viz., BARI Khesari-1, BARI Khesari-2 and BARI Khesari-3, were studied cytogenetically and at the molecular level by using RAPD for genomic characterization. The three varieties were found to posses 2n=14 chromosomes. The karyotype formulae of BARI Khesari-1 and BARI Khesari-2 are 8m + 6sm, while it is 10 m + 4sm in BARI Khesari-3. After orcein and CMA staining, one to two very small chromosome-like bodies were found in some cells of BARI Khesari-1, whereas no such body was observed after DAPI staining. Due to their unique features these can be considered as GC-rich heterochromatic “B-chromosomes.” In BARI Khesari-2, two pairs of satellites were found, while only one pair was present in BARI Khesari-3. The inverted position of satellited regions indicated the probable occurrence of homozygous inversion. An indication of paracentric inversion regarding DAPI-banding pattern was found in pair V of BARI Khesari-2 and thus could be considered as marker chromosomes for this variety. The three varieties have distinct CMA- and DAPI-banding patterns. In BARI Khesari-2, a total of 12 C-bands were found on 10 chromosomes (out of 14 chromosomes). On the basis of band position and length, a deep correlation between C-banding and DAPI-banding pattern was found suggesting the heterochromatic nature of DAPI bands. Each variety showed different RAPD fingerprinting with 59.09% polymorphisim. In addition, a number of variety-specific unique RAPD bands was observed. Therefore, each variety could be characterized on the basis of karyotype and RAPD analysis.