Prodiamine is a mitotic inhibiting herbicide regularly used to control annual bluegrass PRE. A population of annual bluegrass not controlled by prodiamine at 1,120 g a.i. ha−1 was identified on a golf course in Alcoa, TN, in 2012. A whole-plant hydroponics bioassay was used to screen this biotype for prodiamine resistance (PR) compared with a known susceptible population (SS). Multitiller (i.e., > 4 tillers) PR and SS annual bluegrass plants were established in hydroponic culture and exposed to 0, 0.001, 0.01, 0.10, 1.0, and 10.0 mM prodiamine. Exposure to prodiamine at 0.001 mM reduced root growth of the SS biotype to 26% of the nontreated check (i.e., 0 mM prodiamine) but had no effect on the PR biotype. When exposed to 10 mM prodiamine, root growth of the PR biotype was reduced to 24% of the nontreated check compared with 9% for the SS biotype. I50 values for the PR and SS biotypes were 0.04 and 2.8 × 10−6 mM prodiamine, respectively. The PR biotype measured lower in plant height and leaf width than the SS population. In field trials, prodiamine at 560, 840, 1,120, and 1,400 g ha−1 only controlled the PR biotype 0 to 22%. PRE applications of the cellulose biosynthesis inhibitor indaziflam at 35, 52.5, and 70 g a.i. ha−1 controlled this PR biotype 70 to 97%. This marks the second instance of annual bluegrass developing resistance to prodiamine in Tennessee during the past 5 yr. Future research should evaluate indaziflam efficacy for control of other prodiamine-resistant biotypes of annual bluegrass as well as annual bluegrass biotypes resistant to herbicidal inhibitors of 5-enolpyruvylshikimic acid-3-phosphate synthase, acetolactate synthase, and photosystem II.