The mitotic-inhibiting herbicide pronamide controls susceptible annual bluegrass (Poa annua L.) pre- and post-emergence, but in some resistant populations, post-emergence activity is compromised, hypothetically due to a target-site mutation, lack of root uptake, or an unknown resistance mechanism. Three suspected pronamide-resistant (LH-R, SC-R, and SL-R) and two pronamide-susceptible (BS-S and HH-S) populations were collected from Mississippi golf courses. Dose-response experiments were conducted to confirm and quantify pronamide resistance, as well as resistance to flazasulfuron and simazine. Target-sites known to confer resistance to mitotic-inhibiting herbicides were sequenced, as were target-sites for herbicides inhibiting acetolactate synthase (ALS) and photosystem II (PSII). Pronamide absorption, and translocation was investigated following foliar and soil applications. Dose-response experiments confirmed pronamide resistance of LH-R, SC-R, and SL-R populations, as well as instances of multiple resistance to ALS and PSII inhibiting herbicides. Sequencing of the α-tubulin gene confirmed the presence of a mutation that substituted isoleucine for threonine at position 239 (Thr239-Ile) in LH-R, SC-R, SL-R, and BS-S populations. Foliar application experiments failed to identify differences in pronamide absorption and translocation between the five populations, regardless of harvest time. All populations had limited basipetal translocation—only 3–13% of the absorbed pronamide—across harvest times. Soil application experiments revealed that pronamide translocation was similar between SC-R, SL-R, and both susceptible populations across harvest times. The LH-R population translocated less soil-applied pronamide than susceptible populations, 24, 72, and 168 HAT, suggesting that reduced acropetal translocation may contribute to pronamide resistance. This study reports three new pronamide-resistant populations, two of which are resistant to two modes-of-action (MOA), and one of which is resistant to three MOA. Results suggest that both target-site- and translocation-based mechanisms may be associated with pronamide resistance. Further research is needed to confirm the link between pronamide resistance and the Thr239-Ile mutation of the α-tubulin gene.