Objective: In this prospective study, the diagnostic performance of the new version of superb microvascular imaging (SMI) in differentiating malignant from benign lesions was evaluated. Material and Methods: Ninety breast lesions were included. During color SMI examination, both free-hand region of interest (ROI) and box ROI were used. Vascular index (VI) values were obtained from the lesion using both types of ROI and from normal breast tissue via box ROI. VI values, monochrome SMI grading and histopathological results were compared. The efficacy of color SMI and monochrome SMI was investigated in differentiating between benign and malignant breast lesions. Results: The cut-off value, in the differentiation of benign and malignant lesions with color SMI was 0.50 for box ROI, while it was 0.30 for free-hand ROI. The specificity of VI values obtained with box ROI was higher than that of free-hand ROI when differentiating malignant lesions from benign. Comparison of VI values from a lesion and from normal breast tissue showed that VI values in malignant lesions were significantly higher (p<0.05). The VI values of benign lesions and VI values of normal breast tissue were similar. There was a statistically significant relationship between monochrome SMI grading and the malignancy or benign status of the lesion (p<0.001). Conclusion: Drawing the lesion circumference free-hand using a free-shape ROI did not enhance the sensitivity and specificity. Contrary to popular belief, a more easy and practical measurement method may be more suitable for SMI examination. It is hoped that this will be one of the earliest studies to assess the clinical performance of the latest version of SMI.