Quantifying the level of ecophysiological, biochemical, and agronomical fitness of herbicide-resistant (R) and herbicide-susceptible (S) weeds is useful for understanding the evolutionary development of herbicide resistance, but also for implementing herbicide-resistance management strategies. Although germination is a key fitness component in the life cycle of weeds, germinability of S and R weeds has rarely been evaluated under stressful conditions. Germinability traits of S and non–target site resistant subpopulations of blackgrass (Alopecurus myosuroides Huds.) sharing closely related genetic background were tested under salinity, drought stress, and accelerated seed-aging (i.e., exposed to 100% relative humidity at 45 C from 0 to 134 h) conditions. In addition, the activity of three antioxidant enzymes and protein concentration of accelerated aged seeds of the subpopulations were studied. There were no differences in maximum seed germination (Gmax) and time to 50% germination between the two subpopulations under optimum conditions. However, under salinity, drought stress, and accelerated aging conditions, there were differences between the subpopulations. The salinity, drought, and accelerated aging treatments reducing Gmax of the S subpopulation by 50% were 18 dS m−1, 0.75 MPa, and 90 h, respectively, while for the R subpopulation the corresponding values were 15 dS m−1, 0.66 MPa, and 67 h. No differences were found in the activity of the antioxidant enzymes and the content of protein between non-aged seeds of the subpopulations. The aging treatments reducing the activity of catalase and superoxide dismutase enzymes by 50% were 118 and 82 h for the S subpopulation, respectively, while they were 54 and 58 h for the R subpopulation. In contrast, there were no differences in the effect of the aging treatments on the peroxidase activity and protein content between subpopulations. The results provided clear evidence that the non–target site resistant loci of blackgrass is associated with fitness costs under environmental stress.