Common waterhemp is an obligate outcrosser that has high genetic variability. However, under selection pressure, this weed shows population differentiation for adaptive traits. Intraspecific variation for herbicide resistance has been studied, but no studies have been conducted to determine the existence of variation for other adaptive traits that could influence weed management. The objective of this study was to examine the existence of different seed dormancy regulatory mechanisms in common waterhemp. Seed dormancy regulation, in response to different temperature and moisture regimes, was studied through germination experiments and proteome analysis using two common waterhemp biotypes (Ames and Everly) collected from agricultural fields in Iowa, and one biotype (Ohio) collected from a pristine area in Ohio. Without stratification, germination percentage among the different biotypes was 9, 29 and 88% for Ames, Everly, and Ohio respectively. The germination rate of seeds from Ames was dramatically increased after incubation at either 4 or 25 C under wet conditions, whereas germination of seeds from Everly was only increased at 25 C under wet conditions. The Ohio biotype showed no change in germination response to any of the incubation treatments. Germination studies indicated that the rate of seed dormancy alleviation differed between biotypes. Seed protein profiles obtained from the three biotypes differed in protein abundance, number, and type. A putative small heat-shock protein (sHSP) of 17.6 kDa and isoelectric point (pI) 6.1 increased whereas a putative glyceraldehyde-3-phosphate dehydrogenase (G3PDH) of 30.9 kDa and pI 6.4 decreased in abundance in the Ames biotype as seed dormancy was reduced in response to incubation at 4 C and wet conditions. These two proteins did not change in the Everly and Ohio biotypes, suggesting that these proteins changed their abundance in response to seed dormancy alleviation. The results of this study suggest that differences in seed dormancy levels between the biotypes were due to different physiological regulatory mechanisms.