Root rot, caused by Pythium aphanidermatum, is a major disease of sugar beet in warm regions with high levels of soil moisture. Use of resistant cultivars is an effective strategy to control this destructive disease. However, the mechanisms involved in resistance of sugar beet to the pathogen is still unknown. In this research, 10 sugar beet lines were evaluated in greenhouse for resistance to P. aphanidermatum at 6 and 12 weeks growth stages. Our results revealed higher level of resistance in the mature plants. Also, there is no interaction between growth stages and sugar beet lines to P. aphanidermatum suggested the possibility of evaluating resistance of sugar beet genotypes to this oomycetous pathogen at earlier growth stages, which leads to save time and expenses. Activity of defense-related enzymes, including peroxidase (POX), catalase (CAT), polyphenol oxidase (PPO), and ascorbate peroxidase (APX) in the resistant (S2–24-P.107) and susceptible (SB-191) lines inoculated with the pathogen at two different growth stages viz. 6 and 12 week, was evaluated. Obtained results revealed that the activity of POX, CAT, and PPO enzymes was enhanced earlier and to the higher extent in the resistant compared to the susceptible line at both growth stages. The activity of APX was not significantly affected by plant inoculation. Phenolic content of the resistant line was higher than that of the susceptible line at both growth stages investigated. Therefore, our results indicated the prominence of POX, CAT, PPO, and phenolics in sugar beet resistance against P. aphanidermatum.