Accurate identification of contaminant exposure to nontarget organisms involves understanding sources of variation associated with contaminant specific biomarkers. Cholinesterase (ChE) enzyme activity is the biochemical endpoint most often used to assess organism exposure to organophosphorus (OP) pesticides. A potential source of variation for this biomarker is sex, however, a generalized pattern of sex as a source of variation for avian ChE levels remains equivocal. In this study, sex-dependent variation in plasma ChE activity was examined in breeding northern cardinals (Cardinalis cardinalis) from reference populations free of OP pesticide exposure. Male (n = 32) total ChE activity (1.745 +/- 0.541) was greater than female (n = 26) total ChE activity (1.480 +/- 0.576) (p = 0.015). Likewise, benchmarks based on reference means and used for exposure identification (e.g., 20% reduction and the diagnostic threshold) were higher in males than females. With females having a 15.2% lower ChE activity than males, a substantial source of error exists for exposure assessment since some researchers consider a 20% inhibition as evidence of exposure. A second objective was to survey published literature to examine the extent that studies have accounted or controlled for the potential effect of sex on ChE activity. More studies (56.3%) pooled data for male and female ChE activity than studies that statistically tested for an effect (20.3%) or tested hypotheses using a single sex (23.4%) (p < 0.0001; n = 64). Based on these findings, it is suggested that the effect of sex be considered in future studies using the ChE biomarker for identification of passerine exposure to OP pesticides, particularly if examining plasma sources of ChE in actively breeding passerines.