The analysis of Sudan dyes has gained considerable attention during last years. In several publications multiple peaks in chromatograms of Sudan III and Sudan IV have been detected. It is demonstrated in this work that if the sample is kept in darkness before analysis, only one chromatographic peak appears for Sudan III or Sudan IV. In normal lighting conditions two peaks are observed. This light-induced effect can lead to under- or overestimation of the Sudan III and Sudan IV content by 10% and false-positive detection of Sudan I and Sudan II, if proper care is not taken. Appearance of the second peak in chromatograms of Sudan III and Sudan IV is attributed to photochromic E-Z isomerization of these dyes.