Abstract Objectives The TALLYHO (TH) mouse is a polygenic model for obesity, type 2 diabetes and hyperlipidemia. We previously established a congenic mouse strain with TH donor segment on chromosome (Chr) 1 in a C57BL/6 J (B6) background that harbors quantitative trait loci (QTL) conferring obesity and hypercholesterolemia (The B6.TH-Chr1–128Mb). The congenic mouse developed increased adiposity and hypercholesterolemia and demonstrated that distal segment of Chr 1 from TH genome is necessary to cause both phenotypes. With the goal to identify the underlying genes regulating body fat and plasma cholesterol levels on mouse Chr 1, in this study we generated and characterized subcongenic lines with smaller TH donor segments. Methods Using the original B6.TH-Chr1–128Mb congenic mouse, we fixed three overlapping subcongenic lines over the distal region of Chr 1; one containing the full distal region of TH donor genome (39Mb in size) (B6.TH-Chr1–39Mb) and two nested overlapping lines (B6.TH-Chr1–30Mb and B6.TH-Chr1–20Mb). For mapping, all three lines of subcongenic mice and control B6 mice were weaned onto either chow or high fat (HF) diets at 4 weeks of age, and maintained on the respective diets. At 14 weeks of age, body composition including fat and lean mass was measured using EchoMRI-100 whole body composition analyzer, and plasma total cholesterol levels were determined using colorimetric assays. Data were analyzed by ANOVA using GraphPad Prism 8. Results On chow, body fat mass was not significantly different among all subcongenic mice and B6 mice. On HF diets, on the other hand, all three subcongenic lines of mice had significantly larger fat mass than B6 mice without genotype differences. These results suggest that the obesity candidate region is most likely located within the unique segment retained in B6.TH-Chr1–20Mb mice, delimited from the markers D1Mit105 and D1Mit361. Plasma total cholesterol levels were also significantly higher in B6.TH-Chr1–20Mb mice than them in B6 mice both on chow and HF diets. Conclusions We have created subcongenic lines, with smaller TH donor segments from the Chr 1 congenic interval, which develop detectable phenotypes, thus narrowing target intervals for positional cloning. Funding Sources This work was supported, in part, AHA 18AIREA33960437, NIH 1 R15 DK113604–01A1.