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The number of poisoning cases caused by the Lepiota genus is globally increasing. This genus has more poisonous species than the Amanita genus, and many Lepiota species can cause severe toxicity and death if ingested. As recognized in the literature, L. castanea is a toxic species containing amatoxin. Although crude analytical methods have shown that L. castanea contains amatoxins, more recent and sensitive analyses suggest otherwise. Toxin concentrations can vary even among the same fungal species due to geographical and climatic differences. Therefore, this confusion can be resolved by analyzing L. castanea toxins from different geographical regions. This study aimed to demonstrate the toxin levels of L. castanea collected from forests in different regions of Turkiye (Istanbul and Kocaeli) using sensitive methods. The collected mushrooms were analyzed for alpha amanitin, beta amanitin, gamma amanitin, amanin, phallacidin, and phalloidin levels using RP-HPLC-UV and LC-ESI-MS/MS methods. L. castanea mushroom was found to be free of amatoxin and phallotoxin. Our study revealed for the first time that L. castanea mushrooms from different geographical regions of Turkiye do not contain amatoxin and phallotoxin. Supporting these findings with new studies from different parts of the world would be appropriate. [Display omitted] • The literature describes Lepiota castanea as a toxic mushroom that contains amatoxin. • Recent studies have revealed that this mushroom does not contain amatoxin. • Even within the same species, mushroom toxin concentrations can vary due to geographical and climatic differences. • No amatoxin or phallotoxin was detected in L. castanea mushrooms grown in Turkey. • Analyses of other L. castanea mushrooms worldwide should support these data. [ABSTRACT FROM AUTHOR]