Addressing global micronutrient deficiencies through biofortification is essential for improving human health. Sorghum, as a drought-tolerant and resilient crop, offers significant potential in this regard. This study investigated the biochemical traits and genetic variability among red sorghum genotypes, focusing on breeding nutritionally enriched varieties. The cross derivatives of sorghum parental varieties viz., CO 32 × Paiyur 2 in F2 and F3 generation were analysed for key biochemical traits, including total carbohydrate, amylose content, total protein, total phenolic content, total flavonoid content, total tannin content, total anthocyanin content, iron and zinc, as well as antioxidant potential. The genotypes in F2 generation exhibited considerable variation with iron content ranging from 772.15 mg/kg to 1094.84 mg/kg and zinc content between 59.05 mg/kg and 764.23 mg/kg, highlighting the potential for biofortification. Total antioxidant activity ranged from 10.63 % to 88.56 %, indicating diverse antioxidant profiles among the genotypes. The F3 generation genotypes showed improved mean values for total carbohydrate averaging 63.54 g/100 g, amylose content of 38.89 % and Fe content of 958.65 mg/kg. Selection pressure led to reduced genetic variability, as evidenced by changes in skewness and kurtosis and significant improvements in micronutrient and antioxidant content in F3 generation materials. High correlation coefficients (≥0.94) for traits such as amylose content, total protein, iron and zinc content suggested strong genetic control and inheritance of the traits. Heritability estimates above 0.50 indicated the potential for consistent genetic gains. These findings provided valuable insights for developing sorghum varieties with enhanced nutritional profiles, contributing to global food security and addressing micronutrient malnutrition.