Chao Li,1 Xiaofen Ma,2 Mengshi Dong,2 Yi Yin,1 Kelei Hua,1 Meng Li,2 Changhong Li,2 Wenfeng Zhan,2 Cheng Li,2,3 Guihua Jiang2 1Department of Medical Imaging, The Affiliated Guangdong No 2 Provincial People’s Hospital of Southern Medical University, The Third Clinical Medical College of Southern Medical University, 2Department of Medical Imaging, 3Department of Renal Transplantation, The Affiliated Guangdong No 2 Provincial People’s Hospital of Southern Medical University, Guangzhou, People’s Republic of China Objective: Investigating functional specialization is crucial for a complete understanding of the neural mechanisms of primary insomnia (PI). Resting-state functional magnetic resonance imaging (fMRI) is a useful tool to explore the functional specialization of PI. However, only a few studies have focused on the functional specialization of PI using resting-state fMRI and results of these studies were far from consistent. Thus, the current study aimed to investigate functional specialization of PI using resting-state fMRI with amplitude of low frequency fluctuations (ALFFs) algorithm. Methods: In this study, 55 PI patients and 44 healthy controls were included. ALFF values were compared between the two groups using two-sample t-test. The relationship of abnormal ALFF values with clinical characteristics and duration of insomnia was investigated using Pearson’s correlation analysis. Results: PI patients showed lower ALFF values in the left orbitofrontal cortex/inferior frontal gyrus, right middle frontal gyrus, left inferior parietal lobule, and bilateral cerebellum posterior lobes, while higher ALFF values in the right middle/inferior temporal that extended to the right occipital lobe. In addition, we found that the duration of PI negatively correlated with ALFF values in the left orbitofrontal cortex/inferior frontal gyrus, and the Pittsburgh Sleep Quality Index score negatively correlated with ALFF values in the left inferior parietal lobule. Conclusion: The present study added information to limited studies on functional specialization and provided evidence for hyperarousal hypothesis in PI. Keywords: primary insomnia, amplitude of low frequency fluctuations, resting-state fMRI, spontaneous brain activity