The Amaranthus genus contains numerous agronomic weedy species that are widely distributed across the United States. The seeds of many Amaranthus species are morphologically indistinguishable. The Minnesota Department of Agriculture declared Palmer amaranth (Amaranthus palmeri S. Watson) a prohibited noxious weed seed in 2016. Any Amaranthus spp. seeds that are identified as contaminants during routine seed testing require genetic testing to determine whether A. palmeri is present. This research aimed to validate and optimize the molecular detection of A. palmeri in seed. We refined the DNA extraction from pools of Amaranthus spp. seeds ranging in size from 1 to 100 seeds to improve sample testing throughput. Real-time polymerase chain reaction (qPCR) using primers developed by Murphy and colleagues correctly identified the presence of A. palmeri genetics in 84 samples containing 0, 1, 2, 25, or 50 A. palmeri seeds in samples containing up to 100 seeds. The method specificity was examined using 17 Amaranthus species and 4 hybrids of unknown genetics. The Murphy regular qPCR cycle amplified Watson's amaranth (Amaranthus watsonii Standl.), spleen amaranth (Amaranthus dubius Mart. ex Thell.), and spiny amaranth (Amaranthus spinosus L.), which would result in false-positive calls; however, the fast cycle only identified A. watsonii as a potential false positive. Examination of the Murphy primer binding site revealed an identical sequence for A. palmeri and A. watsonii. Additional markers were evaluated and optimized for use in qPCR to eliminate the risk of a false positive. The additional markers did eliminate the amplification of A. dubius and A. spinosus but did not eliminate the amplification for A. watsonii. Currently, A. watsonii is not known to be distributed outside of its limited native range and is not expected to be encountered in samples.