Channa marulius is a freshwater fish of riverine systems known for its economic and ecological importance that has recently been introduced into the farming system. Antibiotic resistance genes reduce the effectiveness of treatments in both public health and aquaculture, leading to resistant pathogens, economic losses, and significant threats to human health and environmental sustainability. The current study aimed to identify antibiotic resistance (ABR) genes in five selected bacterial species and their prevalence in C. marulius sampled from riverine system in Pakistan. Samples were collected from different organs of 480 fish samples of C. marulius. DNA was isolated and ABR genes were identified in the selected bacteria through PCR amplification. Phylogenetic relationship among selected bacteria was compared by phylogenetic tree of gyrB and 16S rRNA gene. Antimicrobial susceptibility was tested against 14 antibiotic discs. A total of 135 (28%) including 29 (6.0%) E. tarda, 33 (6.9%) E. coli, 31 (6.4%) A. hydrophila, 23 (4.8%) F. columnare, and 19 (3.9%) S. aureus isolates, were retrieved. Phylogenetic tree analysis revealed 100% similarity between S. aureus and F. columnare while 90% among A. hydrophila, E. coli, and E. tarda. Maximum 5.62% occurrence of sul3 gene was recorded in E. tarda, 6.46% of qnrA in F. columnare, 5.42% of bla_TEM in E. tarda, and 6.25% of tetA in E. coli. Finally, it was concluded that introduction of wastewater from different industries into rivers causes emergence of ABR genes in pathogenic bacteria which can increase risk of infection and transfer ABR genes to other bacteria.