The current study reports, for the first time, the development of a new analytical method employing ultrasound assisted-cloud point extraction (UA-CPE) for the extraction of CH3Hg+ and Hg2+ species from fish samples. Detection and quantification of mercury species were performed at 550 nm by spectrophotometry. The analytical variables affecting complex formation and extraction efficiency were extensively evaluated and optimized by univariate method. Due to behave 14-fold more sensitive and selective of thiophene 2,5-dicarboxylic acid (H2TDC) to Hg2+ ions than CH3Hg+ in presence of mixed surfactant, Tween 20 and SDS at pH 5.0, the amounts of free Hg2+ and total Hg were spectrophotometrically established at 550 nm by monitoring Hg2+ in the pretreated- and extracted-fish samples in ultrasonic bath to speed up extraction using diluted acid mixture (1:1:1, v/v, 4 mol L-1 HNO3, 4 mol L-1 HCl, and 0.5 mol L-1 H2O2), before and after pre-oxidation with permanganate in acidic media. The amount of CH3Hg+ was calculated from difference between total Hg and Hg2+ amounts. The UA-CPE method showed to be suitable for the extraction and determination of mercury species in certified reference materials. The results were in a good agreement (with Student's t-test at 95% confidence limit) with the certified values, and the relative standard deviation was lower than 3.2%. The limits of detection have been 0.27 and 1.20 mu g L-1, for Hg2+ from aqueous calibration solutions and matrix-matched calibration solutions spiked before digestion, respectively, while it is 2.43 mu g L-1 for CH3Hg+ from matrix-matched calibration solutions. A significant matrix effect was not observed from comparison of slopes of both calibration curves, so as to represent the sample matrix. The method was applied to fish samples for speciation analysis of Hg2+ and CH3Hg+. In terms of speciation, while total Hg is detected in range of 2.42-32.08 mu g kg(-1), the distribution of mercury in fish were in range of 0.7-11.06 mu g kg(-1) for CH3Hg+ and in range of 1.72-24.56 mu g kg(-1) for Hg2+. (C) 2017 Elsevier B.V. All rights reserved.