Sorghum shoot fly, Atherigona soccata,causes substantial economic losses in sorghum globally. Cultural practices and host plant resistance are effective measures for mitigating the losses caused by sorghum shoot fly. Therefore, we evaluated 32 sorghum genotypes consisting of a set of 10 restorer lines, 10 CMS (cytoplasmic male-sterile) lines and their respective maintainers exhibiting resistance/susceptibility to shoot fly along with resistant and susceptible checks under field conditions. The traits such as leaf glossiness, leaf sheath pigmentation, percentage plants with shoot fly deadhearts and number of shoot fly eggs per plant were used as morphological markers for selecting genotypes with resistance to shoot fly during the rainy and post rainy seasons of 2016 and 2017. The test material was also subjected to biochemical analysis (total soluble sugars, protein and tannin contents), while the leaf surface chemicals were analysed by GC-MS to identify the compounds associated with resistance/susceptibility to shoot fly. The genotypes differed significantly for all the traits, except percentage plants with shoot fly deadhearts during the 2016 rainy season. The genotypes ICSB 458, ICSA/B 467, ICSA/B 487, ICSA/B 14037, IS 18551 and ICSV 93046 exhibited moderate to high levels of resistance to shoot fly based on number of plants with shoot fly deadhearts, plants with shoot fly eggs and total number of shoot fly eggs. The shoot fly resistant genotypes ICSB 84, ICSA/B 467, ICSB 487, ICSB 14024, and IS 18551 had low shoot fly deadheart incidence, higher amounts of condensed tannins, soluble sugars, phenols and lower protein content as compared to the susceptible genotypes. Thirteen unique compounds were identified from leaf surface extracts by GC-MS which were associated with shoot fly resistance/susceptibility. While HPLC analysis revealed that Protocatechuic and coumaric acids were present in most of the sorghum genotypes, but their amounts were significantly greater in resistant as compared to the susceptible ones. The findings of the study highlight the importance of various morphological and biochemical traits conferring resistance to sorghum shoot fly, and these traits can be used as markers to identify shoot fly resistant genotypes for use in breeding programs.