In March 2021, Berkeley, California, became the world's first jurisdiction to implement a healthy checkout policy, which sets nutrition standards for foods and beverages in store checkouts. This healthy checkout ordinance (HCO) has the potential to improve customers' dietary intake if stores comply by increasing the healthfulness of foods and beverages at checkouts. To compare the percentage of checkout products that were HCO compliant and that fell into healthy and unhealthy food and beverage categories before and 1 year after HCO implementation in Berkeley relative to comparison cities. In this cohort study in which Berkeley implemented an HCO and other cities did not, a difference-in-differences analysis was conducted of 76 258 product facings at checkouts of 23 stores in Berkeley and 75 stores in 3 comparison cities in California. Data were collected in February 2021 (approximately 1 month before implementation of the HCO) and 1 year later in February 2022 and analyzed from October 2023 to May 2024. The HCO, which permits only the following products at checkouts in large food stores: nonfood and nonbeverage products, unsweetened beverages, and foods with 5 g or less of added sugars per serving and 200 mg or less of sodium per serving in the following categories: sugar-free gum and mints, fruit, vegetables, nuts, seeds, legumes, yogurt or cheese, and whole grains. A product facing's (1) HCO compliance and (2) category, including healthy compliant categories and unhealthy noncompliant categories, determined using a validated photograph-based tool to assess product characteristics. Of the 76 258 product facings at store checkouts, the percentage that were HCO compliant increased from 53% (4438 of 8425) to 83% (5966 of 7220) in Berkeley, a 63% increase relative to comparison cities (probability ratio [PR], 1.63; 95% CI, 1.41-1.87). The percentage of food and beverage checkout facings that were HCO compliant increased in Berkeley from 29% (1652 of 5639) to 62% (2007 of 3261), a 125% increase relative to comparison cities (PR, 2.25; 95% CI, 1.80-2.82). The percentage of Berkeley food and beverage facings consisting of candy, sugar-sweetened beverages, and other sweets significantly decreased (candy: from 30% [1687 of 5639] to 6% [197 of 3261]; PR, 0.21; 95% CI, 0.10-0.42; sugar-sweetened beverages: from 11% [596 of 5639] to 5% [157 of 3261]; PR, 0.41; 95% CI, 0.23-0.75; other sweets: from 7% [413 of 5639] to 3% [101 of 3261]; PR, 0.37; 95% CI, 0.15-0.88), while the percentage consisting of unsweetened beverages (from 4% [226 of 5639] to 19% [604 of 3261]; PR, 4.76; 95% CI, 2.54-8.91) and healthy foods (from 6% [350 of 5639] to 20% [663 of 3261]; PR, 2.90; 95% CI, 1.79-4.72) significantly increased. This cohort study of the first healthy checkout policy found substantial improvements in the healthfulness of food environments at checkouts 1 year after implementation of the policy. These results suggest that healthy checkout policies have the potential to improve the healthfulness of store checkouts.