Euphorbia heterophylla is a species of weed that was previously controlled by fomesafen, imazamox and glyphosate, but continued use of these herbicides has selected resistant populations from the Rio Grande do Sul (Brazil). One resistant (R) strain and one susceptible (S) strain to fomesafen, imazamox and glyphosate were compared, the latter by recurrent selection. Dose-response tests showed multiple resistance to these herbicides. The required imazamox concentration to inhibit ALS by 50% was approximately 16 times greater in the R population than in the S population. Based on the EPSPS activity results, the R population was 10 fold less sensitive to glyphosate than the S counterpart. In addition, basal EPSPS activity from R plants was 3.3 fold higher than the level detected on S plants. The Proto IX assays showed high resistance to fomesafen in the R population that accumulated less Proto IX than the S population. Malathion assays showed the participation of CytP450 in fomesafen resistance, but a molecular mechanism could also be involved. To our knowledge, this is the first characterisation of multiple resistance to these three groups of herbicides in E. heterophylla in the world.