. Here, we examine epidemiological characteristics, specimen collection practices, and cycle threshold (Ct) values for 10,019 MPXV PCR tests performed at two reference laboratories. Results from both laboratories support public health data showing a high positivity rate in men (>30%) and those ages 30 to 49 (25 to 35%). The overall positivity rate decreased during the study period but remains elevated (;20%). There was a significant difference in Ct values between laboratories (ARUP 23.9 versus UW 25.4) and collection method (22.8 for dry swab versus 24.4 for VTM), likely reflecting slight differences in specimen processing. When multiple specimens were collected for a single individual, the overall result concordance rate was greater than 95%, with less than 1.5% of individuals receiving three or more tests having a single positive result. Compared to the overall positive cohort, individuals with three or more swabs collected and a single positive result had significantly higher Ct values (22.9 versus 35.0). Intriguingly, individuals aged 50 to 59 years old had a significantly different viral load distribution than those found in younger age groups, potentially associated with prior vaccinia virus vaccination. These results provide an early snapshot of testing in the United States during the monkeypox virus outbreak and support restricting the number of swabs collected per individual.