The present study reports detailed and comparative karyomorphological and meiotic analyses in Swertia chirayita, S. nervosa and S. bimaculata of Eastern Himalaya. Chromosome analysis of these three Swertia species showed that the majority of cells were diploid with zygotic chromosome number 2n = 26 and gametic chromosome number n = 13. The mosaic chromosome number counts in root tip cells ranged from 9–24 chromosomes in S. bimaculata, to 11–24 chromosomes in both S. chirayita and S. nervosa. No variation was observed in pollen mother cells (PMCs) during meiosis. The modal karyotypes were determined as 2M + 12m + 10sm + 2m:sm for S. chirayita, 8M + 14m + 2sm + 2m:sm for S. nervosa and 6M + 16m + 2sm + 2M:st for S. bimaculata. Karyomorphological analysis of the three Swertia species have documented some distinct similarities between S. chirayita and S. nervosa in terms of their chromosomal category (small), mean chromosomal length (2.48μm, 2.62 μm), length of total chromatin (32.18μm, 34.14 μm) as well as pattern of secondary constriction (m:sm). DNA fingerprinting profiles of S. chirayita, S. nervosa and S. bimaculata were generated employing a total of 39 RAPD primers of which 14 generated distinct reproducible amplified fragments. A total of 189 unique bands were found, of which 72 were of S. chirayita, 49 of S. nervosa and 68 of S. bimaculata. Thus, the present study also reveals DNA markers that can be utilized for identification of genetic diversity of Swertia species. This is the first detailed chromosome analysis report on Swertia species from Eastern Himalaya. © 2015 Dipartimento di Biologia Evoluzionistica, Università di Firenze.