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Morphological as well as Cytogenetical profiling was accomplished in vulnerable freshwater ornamental fish species, namely Pethia setnai (Indigo barb). The Pethia setnai comes under Order – Cypriniformes and Family- Cyprinidae. This species is generally found in west flowing rivers of western ghats of India. The live specimens of Pethia setnai were collected from the Kalna River in Sindhudurg district for the chromosomal as well as morphological analysis. Kidney and Gill cells from two male and two female fishes were used for the study. The chromosomal analysis was carried out by Conventional staining (Giemsa) technique. The cells showed diploid chromosome number i. e. 2n=52 and reported 92 fundamental number (NF) in both the sexes. Karyologically, it composed of 20 metacentric, 8 sub-metacentric, 12 acrocentric and 12 telocentric chromosomes from 25 metaphasic field analysis. The Karyotype formula for the Pethia setnai is 2n (diploid) 52 = 20m+8sm+12a+12t. The long arm chromosome length (L1), total length of chromosome (LT), Relative length (RL), Centromeric index (CI) of chromosome were calculated. The present investigation is the first report of karyological analysis of species P. setnai.