The objective of this study was to evaluate the use of corpus luteum (CL) color doppler ultrasonography (CD) for early pregnancy diagnosis in Bos taurus beef replacement heifers. Beef heifers (n = 183) from two locations were exposed to a 7-d CO-Synch + CIDR protocol followed by fixed-time artificial insemination (day 0). On days 20 and 22, B-mode and CD ultrasonography were performed to evaluate CL morphometries and blood perfusion, respectively. Heifers were considered non-pregnant when CL area was > 20 mm2 or estimated luteal blood perfusion was 25%. Conventional ultrasonography on day 29 was utilized to determine pregnancy status and considered the gold standard method for pregnancy diagnosis. Pregnant heifers had greater CL diameter, CL area, and CL volume when compared to non-pregnant heifers on days 20 and 22 (P > 0.001). Additionally, percentage of central, peripheral, and total luteal blood perfusion, as well as the respective blood perfusion scores were greater (P > 0.001) in pregnant compared with non-pregnant heifers on both day 20 and 22. Sensitivity, specificity, positive predicted value (PPV), negative predicted value (NPV), and accuracy for CD on day 20 were 100, 70, 86, 100, and 90, respectively. Sensitivity, specificity, PPV, NPV, and accuracy for CD on day 22 were 100, 76, 90, 100, and 92, respectively. Pairwise comparison of receiver operating characteristics curve analysis indicated no differences (P = 0.47) between CD on days 20 and 22 (area under the curve = 0.82 and 0.84, respectively). In conclusion, CD successfully detected most non-pregnant replacement heifers on day 20 and 22, while false negative results were absent (NPV = 100%).