The reliability and diagnostic accuracy of commonly used diagnostic imaging modalities in the classification of lumbosacral transitional vertebrae (LSTV) are poorly known, and comparative studies are scarce. To compare the diagnostic performance of conventional radiography (CR), computed tomography (CT), and magnetic resonance imaging (MRI) in classifying LSTVs. In this retrospective cross-sectional study, a total of 852 patients undergoing lumbar imaging studies using all three modalities were initially assessed for the presence of LSTV using CT scans. In total, 100 patients with LSTV anatomy were identified. Four readers performed blinded and independent evaluations of these 100 patients on each modality, and an experienced fellowship-trained radiologist performed a gold standard read using all three modalities. Inter-reader reliability metrics were analyzed in comparison to the gold standard. Statistical software R (4.2.1) was used for the analyses. We found superior diagnostic efficacy for CT: the sensitivity, specificity, accuracy, and balanced accuracy were 76%, 93%, 77%, and 84%, respectively. For MRI, the metrics were 54%, 88%, 56%, and 68%, and for CR 32%, 85%, 42%, and 59%, respectively. Inter-reader reliability was found to be good for CT (κ = 0.63-0.71) and fair for both CR (κ = 0.16-0.32) and MRI (κ = 0.24-0.56). CT had the highest diagnostic performance in all measured metrics with good inter-reader reliability. MRI and CR showed fairly poor sensitivity and accuracy, and thus consideration should be used when classifying LSTVs with these two modalities.