The selective breeding of laboratory rodents with different anxiety-related traits is the subject of growing interest. The present study compared the effects of the benzodiazepine midazolam in the elevated plus maze (EPM) test of anxiety in two lines of Wistar rats that were selectively bred in our laboratory for either high or low anxiety-like traits based on a contextual freezing conditioning paradigm. After phenotyping anxiety-like traits (i.e., conditioned freezing behavior), Carioca High-Freezing [CHF], Carioca Low-Freezing [CLF]) and control rats were intraperitoneally injected (1.0 ml/kg) with .9 % saline or midazolam (.25, .5, .75, and 1.0 mg/kg) and subjected to the EPM 30 min later. After the saline injection, the CHF and CLF groups exhibited lower and higher open-arm exploration in the EPM, respectively, compared with control rats. These results indicate that anxiety-related traits previously selected from an associative learning paradigm can also be phenotypically expressed in an ethologically based animal model of anxiety. All midazolam doses significantly increased open-arm exploration in both CHF and control animals, but this anxiolytic-like effect in CLF rats was only observed at the lowest dose tested (.25 mg/kg). The present findings indicate that these two breeding lines of rats are a useful model for studying anxiety, and the anxiolytic effect of midazolam depends on genetic variability that is associated with basal reactions to threatening situations.