•This is the largest study of mpox NAAT testing in pediatric patients.•63.1% (12/19) of mpox cases occurred in the < 1-year-old or 17-year-old age groups.•Pediatric specimens are more frequently collected from females and non-genital sites.•The positive predictive value of MPXV detection in children was 90.5%. Pediatric mpox cases comprise less than 0.3% of the total cases reported in the United States during the global 2022 outbreak. As a result, relatively little is known about the epidemiology or performance characteristics of clinical testing in this group. We retrospectively extracted and analyzed results for pediatric mpox specimens tested at a national reference laboratory from July to December 2022. During our study period 13.4% (2,063/15,385) of specimens were from individuals <18 years of age. The positivity rate of pediatric specimens was significantly lower than in adults (1.3% vs 22.3%). The pediatric cohort also consisted of a higher percentage of females (42.7% vs 31.0%) and lower percentage of specimens from genital sources (9.0% vs 19.7%) as compared to adults. In children, specimens were most frequently collected from 1-year-olds (10.1%) and least frequently from 11-year-olds (3.5%). Positivity rates were disproportionately elevated in the less than 1-year-old and 17-year-old age groups (7.8% and 6.4%, respectively). Ct values of positive cases were not statistically different between pediatric and adult cohorts (25.2 vs 22.2, p>0.05). When all pediatric cases with an initial positive mpox result were examined, 5/26 were classified as inconclusive and 2/26 were determined to be false positives. The positive predictive value of monkeypox virus detection was 90.5% (95% CI: 70.4–97.4%) in children. These results highlight important differences between pediatric and adult mpox populations and reinforce the need for clinical correlation when reporting positive results from a low prevalence population.