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Summary.Studies on the evaluation of separation efficacies are of primary interest to forensic analysts for newly available pesticides. In the present work, a method is described for the separation evaluation of three selected organophosphorus fungicides (OPFs) ditalimfos (D), edifenfos (E), and tolclofos-methyl (TM) by reversed-phase high-performance thin-layer chromatography (RP-HPTLC). The method is based on the separation of three OPFs on silica gel 60 RP-18WF 254 plates with varying mobile phase compositions (10:0-6:4) of methanol-water. The relationship between the mobile phase composition and retention parameters (R F and R M ) as well as separation parameters (ΔR F , R S , α, R F α ) was analyzed. The three OPFs are completely separated from each other with ΔR F ≥ 0.04, except for the pair of compounds E-TM in the mobile phase composition 10:0, 7:3, 6.5:3.5, and 6:4. Under the chromatographic conditions used, TM was adsorbed the strongest, followed by E and D, which is indicated by the higher R M values obtained for TM. Peak resolution (R S ) values greater than 1.5 were obtained over the entire range of mobile phase composition, except for the pair of compounds E-TM. The UV apex of maximum absorption was 200 nm for all the three OPFs as measured by multiwavelength scan in the UV range, and higher values of peak area and peak height were obtained at the same wavelength. The present study clearly indicates that the system of RP-HPTLC with mobile phase methanol-water in a volume composition 8:2 (v/v) provides the optimum conditions for the separation of the three OPFs. However, the complete separation of E from TM was not achieved using this system.