The objective of the present field study was to establish the beneficial effects of re-insemination of non-pregnant cows using ultrasonography 20 to 23 days after the artificial insemination. A total of 245 Japanese Black cows were artificially inseminated and early pregnancy diagnosis (EPD) was performed on 92 cows 20 days after insemination, using ultrasonography. The remaining 153 cows were considered as negative controls in which routine rectal palpation was performed for pregnancy diagnosis 45-50 days post-insemination. EPD revealed that eleven of the 92 cows (12%) were infertile due to ovarian abnormalities and were thus excluded from the rest of the study. Forty-eight (59%) of the remaining 81 cows were diagnosed as pregnant, while the other 33 (41%) were diagnosed as non-pregnant. Of these non-pregnant cows, 17 of them received a dose of an analogue of the gonadotropin-releasing hormone (GnRH analogue) and were then timed-inseminated, while the other 16 were observed for estrus signs, and 13 of them (81%) were artificially inseminated. Rates of conception were 35% and 38% in the GnRH and the artificially inseminated groups, respectively (P>0.05). Total pregnancy rate for the EPD group increased significantly (74%) (P<0.01) when compared to the control cows (54%) within the same period. In conclusion, our field study demonstrated that re-insemination of non-pregnant cows following EPD is highly efficacious not only in improving the rate of fertility via reducing inter-insemination and inter-calving intervals, but also aids in the early detection of ovarian disorders.