Objective To examine the impact of the COVID-19 pandemic on first year undergraduate student mental health. Methods As part of the Queen’s University U-Flourish Student Well-Being and Academic Success study, three successive cohorts of students entering undergraduate studies in 2018 (pre-pandemic), 2019 (transitional), and 2020 (during pandemic) completed electronic surveys at entry and completion of first year. Validated self-report measures were used to assess mental health status including symptom levels of anxiety, depression, and insomnia, self-harm and frequency of substance use. Propensity matching and multivariable log-binomial regression were used in comparisons of mental health indicators across the cohorts. Results Clinically significant symptoms of depression, anxiety, insomnia, and self-harm were reported more frequently in the 2020–2021 cohort, coincident with remote learning and pandemic restrictions. In female students, screen positive rates for anxiety and depression, and suicidal ideation increased from about one-third to just under one-half in association with the pandemic (χ2, p < .01), while increases in mental health concerns were less pronounced among males. Among females, increases in clinically significant symptoms over first year appeared greatest during the pandemic year, while striking decreases in alcohol consumption in both females and males were reported in that same year. Studying under pandemic conditions had a negative impact on student well-being, social relationships and school connectedness, quality of learning experience, leisure activities, and optimism about future prospects. Conclusions Mental health concerns including anxiety, depression and sleep problems increased in first year students during the pandemic, especially among females, while alcohol use declined. These findings highlight the negative mental health impact associated with studying under pandemic restrictions involving remote learning and social distancing.