Benzodiazepine tolerance is a well-documented consequence of their chronic use, the mechanism of which is not yet fully understood. To investigate tolerance development in mice, we have treated C57BL/6J male and female mice with 10 mg/kg diazepam once daily for 27 days (study 1) or twice daily for 14 days (study 2). In study 1, the Spontaneous Locomotor Activity (SLA) test captured sedation consistent with the reduction in the total distance travelled by diazepam-treated mice of both genders across time, compared to vehicle-treated mice (two-way ANOVA). These effects did not diminish over time but when the mice were treated with diazepam at the end of the protocol and tested in the Open-Field Test (OFT), significant differences in the total time moving were observed between the mice chronically treated with vehicle versus diazepam, consistent with the tolerance development in the latter group (two-way ANOVA). In study 2, similar results were obtained in the SLA test, but in the OFT, the acute treatment with diazepam of chronically vehicle-treated male mice led to behaviors in line with anxiogenic effects, including faster speed of movement, reduced entry into and time spent at the OFT center (three-way ANOVA), while the total time moving was reduced in mice treated acutely with diazepam in line with sedation. Importantly, these behavioral changes were not observed in female mice chronically treated with vehicle or diazepam. Thus, the gender and duration of treatment appear to be important factors in the development and manifestation of benzodiazepine tolerance in mice.