The sorghum shoot fly, Atherigona soccata (Muscidae: Diptera), represents a significant biotic constraint to sorghum production, leading to considerable yield losses globally. This study aimed to systematically classify sorghum genotypes based on their resistance to A. soccata infestation. A total of 188 genotypes were subjected to rigorous evaluation employing standardized screening methodologies. The analysis revealed substantial variability in resistance levels across the genotypes. Based on damage assessments in field trials, 14 genotypes were selected for further investigation under controlled pot culture conditions. Comprehensive biochemical analyses were conducted on each genotype under both uninfested and infested scenarios. Among the evaluated genotypes, IS 10588 and IS 8380 exhibited high levels of resistance, IS 12787 demonstrated moderate resistance, while TNFS 230 was classified as moderately resistant to A. soccata infestation. Critical morphological and biochemical traits associated with resistance were identified, including trichome density, leaf glossiness, and enzyme activity levels of peroxidase (PO), polyphenol oxidase (PPO), tannins, and phenolic compounds. The study concludes that these morpho-physiological and biochemical characteristics contribute significantly to the resistance mechanisms in sorghum against A. soccata. Thus, these identified genotypes may serve as valuable genetic resources for breeding programs aimed at enhancing resistance to A. soccata in sorghum.