Fluorescence molecular tomography (FMT), which can visualize the distribution of fluorescence biomarkers, has become a novel three-dimensional noninvasive imaging technique for in vivo studies such as tumor detection and lymph node location. However, it remains a challenging problem to achieve satisfactory reconstruction performance of conventional FMT in the first near-infrared window (NIR-I, 700-900nm) because of the severe scattering of NIR-I light. In this study, a promising FMT method for heterogeneous mice was proposed to improve the reconstruction accuracy using the second near-infrared window (NIR-II, 1000-1700nm), where the light scattering significantly reduced compared with NIR-I. The optical properties of NIR-II were analyzed to construct the forward model for NIR-II FMT. Furthermore, to raise the accuracy of solution of the inverse problem, we proposed a novel Gaussian weighted neighborhood fused Lasso (GWNFL) method. Numerical simulation was performed to demonstrate the outperformance of GWNFL compared with other algorithms. Besides, a novel NIR-II/NIR-I dual-modality FMT system was developed to contrast the in vivo reconstruction performance between NIR-II FMT and NIR-I FMT. To compare the reconstruction performance of NIR-II FMT with traditional NIR-I FMT, numerical simulations and in vivo experiments were conducted. Both the simulation and in vivo results showed that NIR-II FMT outperformed NIR-I FMT in terms of location accuracy and spatial overlap index. It is believed that this study could promote the development and biomedical application of NIR-II FMT in the future.