To investigate the daily rhythm of rectal and vaginal temperature, we used six mongrel dogs with a mean body weight of 15±3 kg, aged between 2 and 3 years old. Rectal and vaginal temperatures were recorded every 3 h over 48-h period during three different lighting regimes: natural light/dark (L/D) cycle (sunrise 06:25, sunset 17:05), constant light (L/L) and constant darkness (D/D). A daily rhythm of rectal temperature was observed in both days of monitoring in all experimental conditions. Vaginal temperature showed a daily rhythmicity in L/D and D/D cycle. During the L/L cycle, daily rhythm was disrupted. Rhythmic parameters (MESOR, amplitude, acrophase and robustness) did not change between the different photoperiod and the site of temperature collection. In conclusion, the monitoring of vaginal temperature can be considered a valid alternative to the monitoring of body temperature as well as rectal temperature under natural lighting conditions in canine medicine.