Even though mice are being increasingly used as models for human cardiovascular diseases, non-invasive monitoring of cardiovascular parameters such as cardiac output (CO) in this species is challenging. In most cases, the effects of gender and body weight (BW) on these parameters have not been studied. The objective of this study was to provide normal reference values for CO in C57BL/6 mice, and to describe possible gender and/or BW associated differences between them. We used 30-MHz transthoracic Doppler ultrasound to measure hemodynamic parameters in the ascending aorta [heart rate (HR), stroke volume (SV), stroke index (SI), CO, and cardiac index (CI)] in ten anesthetized mice of either sex. No differences were found for HR, SV, and CO. Both SI and CI were statistically lower in males. However, after normalization for BW, these differences disappeared. These results suggest that if comparisons of cardiovascular parameters are to be made between male and female mice, values should be standardized for BW.