Abstract Two resistant (R) biotypes of Setaria faberi and S. viridis have been detected in maize fields continuously treated with triazine herbicides. The R biotypes of S. faberi and S. viridis were 10.0 and 6.5 times, respectively, more resistant to atrazine than their susceptible (S) biotypes. Both R biotype plastids had less affinity for atrazine than the S biotypes based on chlorophyll fluorescence and Hill reaction assays. As expected, they exhibited cross-resistance to a wide variety of photosystem II inhibitors. The hierarchy of resistance level of these two biotypes was chloro- s -triazines ⪢ methoxy- s -triazines > methyltio- s -triazines ≥ as-triazines. There was no difference in the absorption and translocation of [ 14 C]atrazine in R and S biotypes. Fast fluorescence induction curves showed that atrazine inhibited whole-leaf photosynthesis in S biotypes and they later recovered slight fluorescence activity in both populations. All biotypes produced the same pattern of metabolites, but the R biotypes detoxified the atrazine to conjugate-atrazine at a higher rate than the S biotypes (R > 75% > S). The five wild-type Setaria spp. found in Spain showed differences in detoxification of atrazine. The hierarchy of detoxifying level of atrazine of these Setaria spp. was S. verticilata ≃ S. adherens ⪢ S. faberi ≃ S. viridis >⪢ S. glauca .