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This population-based ecological study analyzes the prevalence of childhood leukemia in Ukraine before and after the Chornobyl nuclear power plant accident, based on the contamination status of the territory, time period, gender, and age. Three regions—Zhytomyr, Kyiv (except Kyiv city), and Chernihiv were included as areas contaminated by radioactive 137Cs from 1 to 15 Ci/km2 with annual effective doses exceeding 1.0 mSv, and Sumy region as the control (non-contaminated) area with 137Cs contamination less than 1 Ci/km2 and effective doses less than 0.5 mSv per year. The integrated database of the National Research Centre for Radiation Medicine used in the present study included 1085 childhood leukemia cases. Two aggregated periods were used for analysis: 1980−1986 (pre-accident) and 1987−2000 (post-accident). ICD-9 codes for leukemia (204−208.9) were used to perform analyses according to the extent of leukemic cells maturity (acute, chronic, and maturity unspecified leukemia), leukemic cell lineage (lymphoid, myeloid and lineage unspecified leukemia) and all leukemia cases in different age subgroups (1−4, 5−9, 10−14, and 15−19 years). Standard methods of descriptive epidemiology were used to calculate the prevalence of disease and frequency ratio in regression models. A statistically significant increase in frequency ratio for acute leukemia (1.44; 95% confidence interval (CI), 1.22−1.71), myeloid leukemia (2.93; 95% CI, 1.71−5.40), cell lineage unspecified leukemia (II) (1.48; 95% CI, 1.18−1.87) and all forms of leukemia (1.59; 95% CI, 1.36−1.86) was found for the post-accident period in highly contaminated areas. The results indicate that the frequency of childhood leukemia (and of some of its types) increased in contaminated areas during the post-accident period, suggesting that radiation exposure after the Chornobyl accident might be the cause of the increase. However, further analytical studies, with individual or at least group dose estimates, are needed to confirm a link between childhood leukemia and the Chornobyl accident.