Invasive watermilfoils, specifically Eurasian watermilfoil and the interspecific hybrid of Eurasian watermilfoil × northern watermilfoil, continue to be problematic for water resource managers. Herbicides are often used to control these nuisance weeds and have been historically successful in controlling Eurasian watermilfoil. A population of hybrid watermilfoil from Townline Lake in Michigan has shown increased tolerance to the herbicide fluridone. The objective of this work is to determine if cross- and multiple tolerance have also developed in this population. Eurasian watermilfoil plants collected from multiple sites and plants from Townline Lake were treated with 0, 5, 10, 20, 40, or 80 µg L−1 of fluridone, norflurazon, or topramezone. Fluridone and norflurazon inhibit phytoene desaturase, whereas topramezone is a 4-hydroxyphenylpyruvate dioxygenase-inhibiting herbicide. Chlorophyll fluorescence (Fv/Fm) and pigment content was measured at 10 d after treatment. Townline Lake plants responded differently from susceptible plants when treated with fluridone, norflurazon, and topramezone at 40 µg L−1. These results indicate that the Townline population of hybrid watermilfoil has inherent tolerance to multiple herbicide modes of action. These results are especially significant as topramezone has recently been labeled for aquatic use. Screening of additional herbicides to determine potential herbicide tolerance of the Townline Lake population is recommended.