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        Free- living  amoebae (FLA) are predatory heterotrophic protozoa that are widely distributed  in various environmental sources .They are known to cause serious human infections including a fatal encephalitis, pneumonia and a blinding  keratitis.      was performed in Thi –Qar province south of  Iraq in order to detection of the opportunistic FLA and identified  by morphological characters in the culture , Polymers Chain Reaction (PCR) amplification a targeting specific genes for each genus   During the period from February to September 2020, one hundred and two (102)samples were collected including 75samples from different environmental  sources,  soil , water ( rivers , tap water  , tank water, stagnant water, the marshes water  and  drops of water from the air conditioner  units )  and animals wastes(lizard , birds and wild mice) ,and 27 clinical samples  ( eye, skin, ear, and Cerebral Spinal Fluid)collected from Al-Hussain  teaching hospital , Bint AL-Huda teaching hospital , Al-Hboobi teaching hospital and private laboratories in Thi-Qar province .     All samples were cultured in the Non Nutrient agar medium (NN-agar medium) and then examined by light microscope and recognized morphologically the trophozoites and cysts of  opportunistic amoebas that belong to the genera ; Naegleria  fowleri, Acanthamoeba spp.(A. triangularis ,A. astronyxis ,A. castellini A. polyphaga ), Sappinia spp.(S. pedata , S. diploidea) and Balamuthia mandrillaris, the study found  62 (60.78%)  samples were positive for total opportunistic FLA, and 40(39.21%) samples were negative, the incidence of FLA in environmental samples  were 76% and in clinical samples were18.51 % .