Three studies were conducted to develop pollen tests for the screening of acetyl coenzyme-A carboxylase (ACCase) target-site resistance in a biotype of johnsongrass. The assays were based on germination of johnsongrass pollen in media supplemented with clethodim. Two different methods were used to evaluate pollen germination—a visual assessment and a spectrophotometric assay. The response of pollen to the germination media was linear for 16 h. At 6 h after treatment, absorbance at 500 nm was nearly 0.5; consequently, 6 h was chosen to conduct the pollen assays using the spectrophotometer. Both assessment methods differentiated the susceptible (S) and resistant (R) biotypes. Pollen from the susceptible biotype of johnsongrass was strongly inhibited by increasing concentrations of clethodim, with a GR50 of 25.8 ± 0.6 (SE) µM and GR50 of 16.4 ± 1.7 (SE) µM clethodim by visual assessment and spectrophotometric assessment, respectively. Minimum R/S values were > 3.9 by visual assessment and > 6.1 by spectrophotometric assessment. ACCase target-site resistance is expressed in johnsongrass pollen.