Arsenic speciation in fish was performed using isocratic elution with cation exchange column high-performance liquid chromatography and inductively coupled plasma-mass spectrometry detection. The separation of arsenobetaine and dimethylarsinic acid was performed in 12 min. DOLT-4 dogfish liver and NIST RM-50 albacore tuna certified reference materials were used to evaluate the accuracy for the determination of total arsenic in fish. In addition, reference materials were analyzed for arsenobetaine and dimethylarsinic acid, although they were not certified for these analytes. The limits of detection for arsenic in arsenobetaine and dimethylarsinic acid were 0.20 and 0.33 ng/mL, and the limits of quantitation were 0.58 and 1.11 ng/mL, respectively. Total arsenic and arsenic species were determined in fish using the developed method. The predominant arsenic species was arsenobetaine; the measured concentrations were from 0.43 to 12.0 mg/kg arsenic as arsenobetaine. Dimethylarsinic acid concentrations were below the limit of quantitation. The relative standard deviations were between 3.36 and 10.49 for total arsenic and 3.51 and 9.30 for arsenobetaine.