Guava (Psidium guajava L.) is an important fruit crop in the tropical and sub-tropical world. The morphological, biochemical, and molecular (using 56 microsatellite markers) characterization was carried out on 18 guava genotypes and related species (P. guineense) during 2018–2022. The present study exhibited a wider range of variability: fruit weight (61.5–68.4 g); fruit diameter (4.20–7.91 cm); total soluble solids (8.50–13.40 °Brix); titrable acidity (0.20–0.64%); reducing sugar (2.55–7.00 mg glucose /g); ascorbic acid (76.4–196.3 mg/100 g); total phenol (125.9–305.4 mg GAE/100 g pulp); and total flavonoid content (172.30–948.5 mg QE/100 g pulp). The antioxidant activities (FRAP and DPPH) showed a favorable relationship with ascorbic acid, total phenol, total flavonoid, and total flavonol content. Red-fleshed genotypes were found superior for all the quality parameters over white-fleshed genotypes. The molecular analysis generated 213 alleles from 56 markers, with 2–9 alleles per locus (mean = 3.80). Apart from the genetic diversity, the improved cultivars were also distinguished by a set of markers: RCGH-4 (mPGCIR-184 and mPGCIR-194); RCGH-1 (mPGCIR-108 and mPGCIR-243); RCG-11 (mPGCIR-206 and mPGCIR-325); and RCGH-7 (mPGCIR-16 and mPGCIR-19). The cluster analysis indicated that P. guineense was the most diverse of the cultivated species, and all the red flesh genotypes were close to each other. Furthermore, RCG-11 had a lower seed content (58.89 per 100 g pulp), while RCGH-1 and RCGH-4 were stable for fruit weight and seed number, which can be promoted for commercial production and future crop improvement programs.