Livers from wild pufferfish, Takifugu rubripes, can be described as having a smooth frontal side and an upper-region that is attached to the hepatic portal vein. Based on this description, the liver can be divided into 10 parts (L1-5 and R1-5), and in this work, the lethal potency of each part was determined by mouse bioassay. Among the raw livers from 58 individuals, all 10 parts of 16 individuals, and some parts of 4 individuals showed mouse lethality, but no toxicity was detected in any part of the liver from 22 individuals. In the livers of 4 individuals that were partially toxic, the lethal potency of the toxic parts was less than 4 mouse units (MU)/g, and no part showed especially high toxicity. The remaining 16 individuals were considered non-toxic based on the assay of only one part. Among 13 frozen livers, all parts of 9 individuals were toxic, while all parts of 4 individuals were non-toxic. Liquid chromatography-mass spectrometry analysis revealed that all parts of a weakly toxic raw liver and a strongly toxic frozen liver had tetrodotoxin as the major toxin. Regarding the 16 raw and 9 frozen livers, whose parts were all toxic, the relative lethal potency of each part to the mean lethal potency of the individual (8.9-709 MU/g) was calculated, and subjected to a two-way analysis of variance with 2 factors (left/right and top/bottom) for each group of livers (raw or frozen). The analysis indicated non-significance among factors and interactions at a significance level of 5% in the frozen livers. However, in the raw livers, although no interaction between the 2 factors was detected, the right-side and the 4th-portion from the top were significantly higher than the left-side and the other portions, respectively. Therefore, we concluded that individual inspection using R4, which is the region that lies below the right-center location of the liver, is suitable for toxicity evaluation of liver to ensure the safe consumption of pufferfish.