Physio-biochemical changes in plants is a principal practice to manage harvesting time. The present study was aimed to investigate the effect of flowering stages (10%, 50%, and 100% flowering) on physiological and biochemical properties in four cultivars (Fasta, Hamadani, Meldor, and Sovrana) of third and fourth-year-old plants of Alfalfa (Medicago sativa L.) based on completely randomized design (CRD). The results showed that total phenolic content (TPC) and total flavonoid content (TFC) in Hamadani cultivar at middle flowering stages in fourth-year-old plants showed higher TPC and TFC. The catalase (CAT) and superoxide dismutase (SOD) activities increased by progressing the flowering stage and time of harvesting. Proline content increased by advancing the flowering stage as its highest amount was observed at late flowering stage in Fasta cultivar. Middle flowering stage represented the greater chlorophyll (Chl.) compared with other stages. Relative water content (RWC) decreased by progressing the maturity as its highest amount was observed at early flowering stage. RWC differed from 65% in fourth-year plants of sovrana at late flowering stage to 80% in third-year plants of Meldor at early flowering stage. Malondialdehyde (MDA) content and total soluble sugar (TSS) increased by advancing the maturity. Sovrana at late flowering stage and fourth year represented the higher MDA and TSS. Heat map analysis showed the significant role of all physiological and biochemical traits except total Chl. for clustering the cultivars. This work suggests the Hamadani cultivar because of high phenolic compounds and the minimum variation of their biochemical properties in the fourth year. پرونده مقاله