Ability to visualize breast lesion vascularity and quantify the vascular heterogeneity using contrast-enhanced three-dimensional (3D) harmonic (HI) and subharmonic (SHI) ultrasound imaging was investigated in a clinical population. Patients (n = 134) identified with breast lesions on mammography were scanned using power Doppler imaging (PDI), contrast-enhanced 3D HI and 3D SHI on a modified Logiq 9 scanner (GE Healthcare). Ultrasound contrast agent (UCA) flow was identified in 4D View™ (GE Medical Systems) and used to generate a map of time-intensity curves for each lesion volume. Time-points corresponding to baseline, peak intensity and washout of UCA were identified to develop vascular heterogeneity plots. Vascularity was observed with PDI in 82 lesions (61 benign and 21 malignant). 3D HI showed flow in 8 lesions (5 benign and 3 malignant), while 3D SHI visualized flow in 68 (49 benign and 19 malignant). Analysis of vascular heterogeneity in the 3D SHI volumes found benign lesions having a significant difference in vascularity between central and peripheral sections (1.71 ± 0.96 vs. 1.13 ± 0.79 dB, p < 0.001, respectively) whereas malignant lesions showed no difference (1.66 ± 1.39 vs. 1.24 ± 1.14 dB, p = 0.24), indicative of more vascular coverage. These preliminary results suggest quantitative evaluation of vascular heterogeneity in breast lesions using contrast-enhanced 3D SHI is feasible and able to detect variations in vascularity between central and peripheral sections for benign and malignant lesions. Finally, 3D parametric volumes were produced for perfusion (PER) and area under the curve (AUC).