Rice (Oryza sativa L.) agriculture of the southern United States is plagued by strong biotic competition with several species in the Echinochloa genus. Despite clear genomic differences between barnyardgrass (Echinochloa crus-galli (L.) P. Beauv.] and junglerice (Echinochloa colona (L.) Link], the two major Echinochloa agricultural weeds are nearly indistinguishable phenotypically. This inability to reliably differentiate the species has led farmers to treat the group as a single species, often resulting in ineffective weed control efforts. In this study, we first develop a simple chloroplast-anchored PCR-based restriction enzyme assay to differentiate between E. colona and the other Echinochloa species of agricultural concern. Applying this assay, we identify a strong bias towards E. colona in 2024 rice field collections from eastern Arkansas. Finally, we evaluate anecdotal reports of interspecific hybridization between species and find no evidence. Despite the drawbacks of the maternally inherited nature of the chloroplast, the availability of this species determinant assay will help USDA and academia extension agents and stakeholders to make educated, species-specific decisions about precision chemical weed control and field management.