PURPOSE: To validate the use of harmonic ultrasonography (US) in the detection of gallbladder microlithiasis. METHODS: From November 30, 2012, to January 18, 2014, fundamental US (FUS) and harmonic US with a high background noise (HUS-N) were performed for evaluation of gallbladder during the routine abdomen US. During the US, a dot-like stone (or stones) with Brownian motion was regarded as a positive finding of microlithiasis. Fifty-five patients with microlithiasis in the gallbladder detected on US were enrolled as the subjects of a retrospective review. With respect to the obtained images, two abdominal radiologists independently scored the conspicuity of gallbladder microlithiasis on FUS and HUS-N by using a 4-grade scale. The statistical analysis employed a kappa test and a Wilcoxon rank-sum test. RESULTS: For FUS, the conspicuity grades of gallbladder microlithiasis were G1 in 25 and 37, G2 in 21 and 9, G3 in 6 and 6, G4 in 3 and 3 patients, while HUS-N showed G1 in 0 and 0, G2 in 3 and 2, G3 in 12 and 15, and G4 in 40 and 38 patients, respectively, by each of the two radiologists. The kappa value was 0.633 for FUS between the two radiologists and 0.708 for HUS-N. HUS-N showed better conspicuity of gallbladder microlithiasis than FUS with significant P-values of less than 0.001 and 0.001 for the two radiologists, respectively. CONCLUSION: Compared with FUS, HUS-N enables better detection of microlithiasis in the gallbladder.