Precocity and efficiency of ultrasonography for pregnancy diagnosis and determination of litter size in mice were assessed on a total of 46 adult mice of different lines (19 BALB/c, 15 C57BL/6 and 12 CD1) from Day 4.5 after vaginal plug. Different commercial ultrasound machines and probes (linear versus sectorial; 7.5 MHz versus 10 MHz) were compared. The best images were obtained by the use of 10 MHz linear transducers. The first visualization compatible with pregnancy, specifically with the implantation sites, was observed at Day 4.5 in three animals. Presence of embryonic vesicles was differentiated at Day 5.5 in all the females. The embryos and remaining gestational structures inside the vesicles were clearly distinguished at Day 8.5. Data were validated not only after delivery but also by comparison with postmortem findings on crucial days (Days 4.5 and 8.5). The efficiency for counting the exact number of embryos was very low, due mostly to underestimation in highly prolific females. Conversely, the estimation of the range of the number of conceptuses, instead of the total number, was more accurate. Sensitivity, specificity and total efficiency reached 100% at Day 8.5. Ultrasonography can be accurately used as an alternative non-invasive technique for pregnancy diagnosis and determination of litter size in the mouse from very early stages of gestation, replacing other procedures currently used and increasing efficiency in animal management and research.