Purpose To retrospectively define the strength of association between testicular microlithiasis and testicular neoplasia in a large geographically diverse pediatric population. Materials and Methods Retrospective review of scrotal ultrasonographic (US) examination reports and pathology specimens obtained between January 2000 and May 2014 at six academic pediatric hospitals in North America was performed. Reported cases were reviewed to confirm microlithiasis. Radiology and pathology data bases were searched for pathology-proven testicular tumors (benign or malignant germ cell or stromal tumors). Association strength (risk) was expressed in terms of odds ratios (ORs) with and without adjustment for fixed study site effects based on logistic regression. Results A total of 37 863 individuals underwent scrotal US during the study period. Mean age was 11.1 years ± 4.7 [standard deviation] in boys with microlithiasis and 9.1 years ± 5.9 in boys without microlithiasis (P < .001). Microlithiasis was confirmed in 2.90% of patients (1097 of 37 863; range, 1.61%-5.25% across sites). It was unilateral in 21.97% (241 of 1097) of patients and bilateral in 78.0% (856 of 1097). Tumor was identified in 4.64% (51 of 1097) of boys with microlithiasis and 0.33% (122 of 36 766) of boys without (unadjusted OR, 14.65; 95% confidence interval [CI]: 10.29, 20.84; adjusted OR, 14.19). Malignant germ cell tumors were identified in 2.8% (31 of 1097) of boys with microlithiasis and 0.12% (45 of 36 766) of boys without microlithiasis (unadjusted OR, 17.26; 95% CI: 11.8, 25.25; adjusted OR, 22.37). Sex cord-stromal tumors were identified in 0.46% (five of 1097) of boys with microlithiasis and 0.079% (29 of 36 766) of boys without (unadjusted OR, 5.8; 95% CI: 2.1, 16; adjusted OR, 6.39). Conclusion There is a strong association between testicular microlithiasis and primary testicular neoplasia in this pediatric population. RSNA, 2017.