Pythium species are common root rot pathogens at hydroponic production systems. While P. aphanidermatum is commonly isolated from roots of plants grown hydroponically, a new species, P. oopapillum, has emerged as an aggressive pathogen of hydroponic spinach and lettuce. The virulence of P. aphanidermatum (lettuce), P. oopapillum 06 (spinach) and P. oopapillum 07 (lettuce) isolates against lettuce plants was evaluated in a simulated deep water hydroponic system. Plants were inoculated by placing potato carrot agar discs with 3-4-day-old mycelial cultures of Pythium isolates in the root zone of the plants. Controls were a water agar disc placed in the root zone or no agar disc. Data were collected for root length, leaf fresh weight, and root fresh and dry weight. There were no statistically significant differences between the non-inoculated controls in any of these parameters. Plants inoculated with isolates of either species had significant reductions in root length, fresh and dry root weight, and leaf weight compared to the control. Plants inoculated with P. aphanidermatum had significantly lower leaf fresh weight than plants inoculated with either isolate of P. oopapillum.