ImportanceInformation about national substance use trends among youths and adults after mid-March 2020 is limited due to constraints on surveillance during the COVID-19 pandemic.ObjectiveTo evaluate whether substance use prevalence in the early part of the pandemic (2020) differed from the prepandemic periods of 2018 to 2019 and 2016 to 2018.Design, Setting, and ParticipantsThis cross-sectional study was a repeated analysis of 2016 to 2020 data from a nationally representative sample of youths and adults in the Population Assessment of Tobacco and Health (PATH) Study. Participants were representative of the US civilian noninstitutionalized population. Household residents age 13 years or older were interviewed in person from 2016 to 2019 and via telephone in 2020.ExposuresAge, calendar year.Main Outcomes and MeasuresPast 30-day self-reported use of any tobacco, any alcohol, binge drinking, cannabis, and any other illegal or misused prescription drugs.ResultsThe overall nationally representative 2020 sample included 7129 youths (ages 13-17 years), 3628 young adults (ages 18-20 years), and 8874 adults (ages ≥21 years). Comparing 2018 to 2019 with 2020 among youths, prevalence of all substances used declined (eg, cannabis use declined in those aged 16-17 years from 14.9% to 7.6%; absolute difference, −7.3 percentage points [95% CI −8.8 to −5.8 percentage points]). Among young adults, prevalence of all substances other than any alcohol decreased significantly (eg, tobacco use declined from 37.8% to 22.8%; absolute difference, −15.1 percentage points [95% CI −16.8 to −13.3 percentage points]). In adults ages 21 to 24 years, any tobacco use declined from 39.0% to 30.9% (absolute difference, −8.2 percentage points [95% CI, −10.6 to −5.7 percentage points]), and alcohol use increased from 60.2% to 65.2% (absolute difference, 5.0 percentage points [95% CI, 2.3 to 7.7 percentage points]). Among adults aged 25 years or older, any tobacco use declined from 39.0% to 30.9% (absolute difference, −8.2 percentage points [95% CI, −10.6 to −5.7 percentage points]), cannabis use increased from 11.3% to 12.4% (absolute difference, 1.2 percentage points [95% CI, 0.3 to 2.0 percentage points]), and other substance use declined from 5.8% to 3.7% (absolute difference, −2.1 percentage points [95% CI, −2.9 to −1.4 percentage points]).Conclusions and RelevanceIn this cross-sectional study, substance use decreased between 2019 and 2020 among those aged 13 to 20 years; consistent declines were not seen in older persons other than tobacco use reductions, and cannabis use increased among adults ages 25 years and older. While social changes during the COVID-19 pandemic could have affected substance use, findings should be interpreted with caution due to differences in data collection methods in 2016 to 2019 and 2020.