ABSTRACT:Mycobacterium arupense is a non-tuberculous, potentially pathogenic species rarely isolated from humans. The aim of the study was to ascertain the spectrum of non-tuberculous mycobacteria within 271 sequenced mycobacterial isolates not belonging to M. tuberculosis and M. avium complexes. Isolates were collected between 2004 and 2009 in the Czech Republic and were examined within the framework of ecological studies carried out in animal populations infected with mycobacteria. A total of thirty-three mycobacterial species were identified. This report describes the isolation of M. arupense from the sputum of three human patients and seven different animal and environmental samples collected in the last six years in the Czech Republic: one isolate from leftover refrigerated organic dog food, two isolates from urine and clay collected from an okapi (Okapia johnstoni) and antelope bongo (Tragelaphus eurycerus) enclosure in a zoological garden, one isolate from the soil in an eagle's nest (Haliaeetus albicilla) band two isolates from two common vole (Microtus arvalis) livers from one cattle farm. All isolates were identified by biochemical tests, morphology and 16S rDNA sequencing. Also, retrospective screening for M. arupense occurrence within the collected isolates is presented.