Abstract: Prominent primary aneuploid stocks namely seven primary trisomics, seven primary tetrasomics, and six double trisomic types were earlier developed in grass pea (Lathyrus sativus L.), a hardy legume. Despite distinct morphological features, identity and nature of their extra chromosome(s) were elusive, hampering assignment of desirable breeding traits into specific linkage groups. The present study aims to analyze the banding pattern and to reveal the identity of extra chromosome(s) involved in these 20 aneuploid types in grass pea. Conventional orcein banding was first done using the root-tip squash technique in all aneuploids along with disomic (2n = 2x = 14) parent, and karyomorphological features were noted. Chromosomes were classified following the total length of individual chromosomes and arranged in order of decreasing sizes, keeping their centromeres in a straight line. DNA-base specific chromomycin A3 (CMA) and 4,6ʹdiamidino-2-phenylindole (DAPI) banding pattern were finally employed to convincingly distinguish the chromosomes from each other. Molecular banding and idiograms revealed unique CMA and DAPI banding pattern in each of the seven chromosome pairs and identified extra chromosome(s) involved in aneuploidy. Fluorescence banding also showed differential CMA-specific GC-rich repeat region and DAPI-specific AT-rich repeat regions in chromosomes with the longest chromosome bearing GC-rich nucleolar organizing region. No polymorphism within homologous pairs was found. The result confirmed the primary nature of present aneuploids and identified the additional chromosome(s) in aneuploids. Based on the extra chromosome(s) involved, new designation of grass pea aneuploids has now been proposed. The results have immense significance in location of desired traits or mutations on specific linkage groups of grass pea. © 2016 Dipartimento di Biologia Evoluzionistica, Università di Firenze.