Ninety-five Escherichia coli isolates recovered from different supply points of freshwater fish namely fish pond (W1, n = 16), wholesale fish market (W2, n = 14), retail fish market (R1, n = 29), street fish vending unit (R2, n = 30), and modern fish vending unit (R3, n = 6) were investigated for antimicrobial-resistant profile and prevalence of antibiotic-resistant genes. Overall, multidrug-resistant (MDR) E. coli in the fish supply chain was 21% with higher MDR incidence in the downstream fish supply points. MDR was relatively higher in the tertiary fish supply points viz., retail fish market (45%), street fish vending unit (30%), and modern fish vending unit (15%) compared to the secondary fish supply point, that is, wholesale fish market (5%) and primary fish supply point, that is, fish pond (5%). All the ampicillin-resistant isolates carried ampC gene, whereas only 21% of the β-lactam resistant isolates carried blaCTX-M-gp1. The tetracycline resistance determinant, tetA (89%) was predominant in the tetracycline-resistant isolates compared to tetB and tetD (11%). The trimethoprim resistance gene, dfrA and sulfonamide-resistance gene, Sul1 were detected in 9% of the trimethoprim-resistant isolates and 9% of the sulfonamide-resistant isolates, respectively. Enterobacterial repetitive intergeneric consensus-PCR has delineated the MDR isolates from the different fish supply points into three major clusters but all the MDR isolates from the street fish vending point are grouped into a single cluster. The results indicate an increase in the proportion of MDR E. coli and the occurrence of diverse MDR profiles in the downstream points of the fish supply chain that needs to be addressed to avoid fish-food-borne antimicrobial resistance.