Obesity is a heterogeneous disorder which increases risks for multiple metabolic diseases, such as type 2 diabetes. The current study aims to characterize and compare visceral and subcutaneous adipose tissues in terms of macromolecular content and investigate transdifferentiation between white and brown adipocytes. Regarding this aim, Fourier transform infrared (FTIR) microspectroscopy and uncoupling protein 1 (UCP1) immunohistological staining were used to investigate gonadal (visceral) and inguinal (subcutaneous) adipose tissues of male Berlin fat mice inbred (BFMI) lines, which are spontaneously obese. The results indicated a remarkable increase in the lipid/protein ratio, accompanied with a decrease of UCP1 protein content which might be due to the transdifferentiation of brown adipocytes to white adipocytes in obese groups. It has been widely reported that brown adipose tissue has a strong effect on fatty acid and glucose homeostasis and it could provide an opportunity for the therapy of obesity. When the amount of brown adipose tissue was decreased, lower unsaturation/saturation ratio, qualitatively longer hydrocarbon acyl chain length of lipids and higher amount of triglycerides were obtained in both adipose tissues of mice lines. The results also revealed that subcutaneous adipose tissue was more prone to obesity-induced structural changes than visceral adipose tissue, which could originate from it possessing a lower amount of brown adipose tissue. The current study clearly revealed the power of FTIR microspectroscopy in the precise determination of obesity-induced structural and functional changes in inguinal and gonadal adipose tissue of mice lines.