Food and water are the main sources of human exposure to arsenic. It is important to determine arsenic species in food because the toxicities of arsenic vary greatly with its chemical speciation. Extensive research has focused on high concentrations of arsenic species in marine organisms. The concentrations of arsenic species in freshwater fish are much lower, and their determination presents analytical challenges. In this review, we summarize the current state of knowledge on arsenic speciation in freshwater fish and discuss challenges and research needs. Fish samples are typically homogenized, and arsenic species are extracted using water/methanol with the assistance of sonication and enzyme treatment. Arsenic species in the extracts are commonly separated using high-performance liquid chromatography (HPLC) and detected using inductively coupled plasma mass spectrometry (ICPMS). Electrospray ionization tandem mass spectrometry, used in combination with HPLC and ICPMS, provides complementary information for the identification and characterization of arsenic species. The methods and perspectives discussed in this review, covering sample preparation, chromatography separation, and mass spectrometry detection, are directed to arsenic speciation in freshwater fish and applicable to studies of other food items. Despite progress made in arsenic speciation analysis, a large fraction of the total arsenic in freshwater fish remains unidentified. It is challenging to identify and quantify arsenic species present in complex sample matrices at very low concentrations. Further research is needed to improve the extraction efficiency, chromatographic resolution, detection sensitivity, and characterization capability.