Cannabis sativa is a versatile crop of immense industrial and medicinal value, cultivated for its fiber, seeds, and especially for its bioactive cannabinoids, yet early identification of sex and chemotype remains challenging due to delayed morphological differentiation and limited cannabinoid accumulation during early growth. This study validated reproducible DNA markers for early selection in Indian cannabis germplasm. For sex determination, i.e., three Sequence Characterized Amplified Region (SCAR) markers-SCAR119, SCAR323, and the Male-Associated DNA Clone 2 (MADC2), were evaluated. The MADC2 marker reliably amplified a 390 bp fragment in male plants, whereas most female plants exhibited 560 bp and 870 bp bands. This strong correlation between MADC2 genotypes and phenotypic sex underscores its utility for early sex identification, which is critical for maximizing cannabinoid yield by preventing unwanted pollination. In parallel, chemotype-specific markers were assessed to predict cannabinoid profiles. Using high-performance liquid chromatography (HPLC), plants were classified into three chemotypes based on THCA: CBDA ratios: THC-dominant, intermediate, and CBD-dominant. The dominant marker D589 was associated with THC biosynthesis, while the co-dominant marker B1080/B1192 provided refined differentiation between chemotypes by detecting allelic variations in cannabinoid synthase genes. Cloning and sequence analysis of these markers revealed significant similarity to known genomic regions involved in cannabinoid production. In summary, the validated SCAR markers enable early and accurate identification of sex and cannabinoid chemotype in Cannabis sativa. These tools offer substantial benefits for marker-assisted breeding, regulatory compliance (e.g., maintaining THC levels below 0.3% for hemp), and optimizing cultivation practices for both industrial and pharmaceutical applications in India.