ABSTRACT American slough grass (Beckmannia syzigachne Steud. Fernald) is an annual grass which has developed resistance to acetyl-coenzyme A carboxylase (ACCase) inhibitors, and the major mechanism of resistance is target site based. Target site resistance-conferring mutations may confer pleiotropic effects on a weed's life cycle. The effects of three American slough grass spontaneous ACCase mutations (Ile1781Leu, Trp2027Cys and Ile2041Asn) on seed germination and viability under different experimental factors had been investigated in this paper. Seeds containing homozygous 1781Leu/Leu ACCase showed a greater germination under salt and osmotic stress, but no fitness variation in optimal experimental conditions. Homozygous 2027Cys/Cys ACCase caused nonsignificant effects on seed germination in optimal experimental conditions, but caused a greater germination after 16 d incubation at 40 °C and 100% RH. Homozygous 2041Asn/Asn ACCase tended to cause nonsignificant effects on seed germination or seed viability. Finally, an obvious population effects on seed germination was observed between six subpopulations, which indicated that fitness assessing should be measured in resistant and susceptible individuals that share a similar genetic background. These findings suggest that the absence of fitness penalty associated with these three homozygous mutant ACCase alleles may be a contributing factor for resistance spread.