The aim was to characterize changes in the ultrasound characteristics of the corpus luteum (CL) and uterus in pregnant, inseminated non-pregnant and cyclic beef heifers and to correlate findings with systemic progesterone (P4) concentrations with the intention of identifying possible markers for early identification of pregnancy. Heifers were randomly selected for artificial insemination (AI) following estrus synchronization. Ultrasound examinations of the CL and uterus were carried out by trans-rectal ultrasonography using a high resolution ultrasound scanner equipped with a 12MHz linear arrayprobe on Days 7, 11, 14, 16 and 18 after AI (Day 0; i.e., estrus). Cross-sectional B-mode images of the CL were captured for calculation of CL tissue area and echotexture. Images of the CL and associated blood flow were captured and stored for analysis of luteal blood flow area and ratio. Longitudinal, B-mode images of the uterine horns were captured just beyond the bifurcation of the uterine horns and stored for analysis of contrast and homogeneity (MaZda v4.6, Technical University of Lodz, Institute of Electronics, Poland). A total of three images were captured for each structure of interest. Serum concentrations of P4 were determined from blood samples collected at each ultrasound examination. Following pregnancy diagnosis by ultrasound, heifers were retrospectively allocated as being pregnant (embryonic heartbeat on Day 28; n=14) or non-pregnant (inter-estrous interval 18 to 21 days; n=8) and their data were compared with non-inseminated, cyclic heifers (n=10). CL tissue area did not appear to change between pregnant, non-pregnant or cyclic control groups between Days 7 to 18 (P>0.05). No significant differences in CL echotexture characteristics were found between groups at any time point. There were no significant differences between pregnant, non-pregnant and cyclic control groups for CL blood flow area (P>0.05). However, CL BF ratio decreased significantly (P<0.05) in both inseminated non-pregnant and cyclic heifers between Days 14 and 18 while it remained unchanged in pregnant heifers (P>0.05). Uterine homogeneity was not significantly different between groups at any time point (P>0.05). However, uterine contrast was significantly higher (P<0.05) in pregnant compared with cyclic control heifers on Days 16 and 18. Concentrations of P4 were lower (P<0.05) in non-pregnant and control heifers than in pregnant heifers from Days 16 to 18. In conclusion, there were differences between non-pregnant and cyclic compared with pregnant heifers in P4 concentrations from Day 16. On Day 18 the CL and uterine characteristics were different between the non-pregnant and pregnant heifers. Ultrasound measures of CL blood flow and uterine echotexture may be useful to establish pregnancy status. Further investigation is required to identify if pregnancy diagnosis can be made on Day 18 or at a later day post-partum.