Glyphosate is now the most widely used herbicide; after years of frequent utilization, resistant weeds were selected, mainly due to widespread adoption of glyphosate-resistant crops and no-tillage sowing system. Increasing difficulty in controlling Chloris polydactyla with glyphosate has been noticed in agricultural areas. Here, the susceptibility level of various C. polydactyla accessions from Brazil is examined. Two whole-plant studies were conducted to confirm the presence and extent of glyphosate susceptibility among accessions, which involved the application of seven glyphosate doses on four accessions. The four accessions showed different glyphosate susceptibilities. The “Matão” accession presented major tolerance for glyphosate compared to “Palotina” accessions. “Jaboticabal” plants showed an intermediary susceptibility. The resistance factor (RF) was 3.76 between the “Matão” and “Palotina” accessions. All biotypes died at 2,880 g ae ha−1 glyphosate.