ObjectivesHere we report on the intra‐ and inter‐operator variability of the backscatter coefficient (BSC) estimated with a new low‐variance quantitative ultrasound (QUS) approach applied to breast lesions in vivo.MethodsRadiofrequency (RF) echo signals were acquired from 29 BIRADS 4 and 5 breast lesions in 2 sequential cohorts following 2 imaging protocols: cohort 1) radial and antiradial views, and cohort 2) short‐ and long‐axis views. Protocol 2 was implemented after retraining and discussion on how to improve reproducibility. Each patient was scanned by at least 2 of 3 radiologists; each performed 3 acquisitions with transducer and patient repositioning in between acquisitions. BSC was estimated using a low‐variance QUS approach based on regularization. Intra‐ and inter‐operator variability of the intra‐lesion median BSC was evaluated with a multifactorial ANOVA test (P‐values) and the intraclass correlation coefficient (ICC).ResultsInter‐operator variability was only significant in the first protocol (P < .007); ICCinter = .77 (95% CI .71–.82), indicating good inter‐operator agreement. In the second protocol, the inter‐operator variability was not significant (P > .05) and agreement was excellent (ICCinter = .92 [.89–.94]). In both protocols, the intra‐operator variability was not significant.ConclusionsOur findings demonstrate the need for standardizing image acquisition protocols for backscatter‐based QUS to reduce inter‐operator variability and ensure its successful translation to the characterization of suspicious breast masses.