Field and greenhouse research was conducted to quantify the level of resistance to atrazine in Wisconsin (WRB1) and Maryland (MRB) velvetleaf biotypes and to determine cross-resistance of the WRB1 and MRB biotypes to other selected herbicides as compared to a Wisconsin atrazine-susceptible velvetleaf accession (WSA1). In field studies, the WRB1 and MRB biotypes survived atrazine applied POST at dosages as high as 4.5 kg ha−1. In contrast, the WSA1 accession had 50% survival following a 1.1 kg ha−1 POST atrazine application. The WRB1 biotype demonstrated neither cross-resistance nor negative cross-resistance to alachlor, bentazon, bromoxynil, cyanazine, dicamba, linuron, metribuzin, or thifensulfuron. The MRB biotype demonstrated neither cross-resistance nor negative cross-resistance to alachlor, bentazon, dicamba, metribuzin, or thifensulfuron; slight negative cross-resistance was demonstrated to bromoxynil, cyanazine, and linuron. In greenhouse studies, the WRB1 and MRB biotypes were approximately 100-fold more resistant to atrazine than the WSA1 accession; the WRB1 and MRB biotypes demonstrated neither cross-resistance nor negative cross-resistance to alachlor, ametryn, bentazon, bromoxynil, cyanazine, dicamba, linuron, metribuzin, pendimethalin, terbacil, or thifensulfuron. Absence of cross-resistance to cyanazine, ametryn, metribuzin, and terbacil in the WRB1 and MRB biotypes of velvetleaf is in contrast to most other atrazine-resistant weed biotypes.