Simple SummarySporadic but repeated occurrences ofSarcinaspp. indicate that these microorganisms with atypical morphology forming packets in the fecal microbiota of animals without health problems may not always be pathogenic and seem to be a common part of the gut microbiota of various mammals. Aside from that, genotyping characterization indicates species and strain variability amongSarcinaisolates and the potential presence of two novel taxonomic units originating from dog and elephant hosts.AbstractSarcinaspp. has been isolated from the gastrointestinal tracts of diverse mammalian hosts. Their presence is often associated with host health complications, as is evident from many previously published medical case reports. However, only a handful of studies have made proper identification. Most other identifications were solely based on typicalSarcina-like morphology without genotyping. Therefore, the aim of this work was culture detection and the taxonomic classification ofSarcinaisolates originating from different mammalian hosts.Sarcina-like colonies were isolated and collected during cultivation analyses of animal fecal samples (n= 197) from primates, dogs, calves of domestic cattle, elephants, and rhinoceroses. The study was carried out on apparently healthy animals kept in zoos or by breeders in the Czech Republic and Slovakia. Selected isolates were identified and compared using 16S rRNA gene sequencing and multi-locus sequence analysis (MLSA;Iles, pheT, pyrG, rplB, rplC, andrpsC). The results indicate the taxonomic variability ofSarcinaisolates.S. ventriculiappears to be a common gut microorganism in various captive primates. In contrast, a random occurrence was also recorded in dogs. However, dog isolate N13/4e could represent the next potential novelSarcinataxonomic unit. Also, a potentially novelSarcinaspecies was found in elephants, with occurrences in all tested hosts.S. maximaisolates were detected rarely, only in rhinoceroses. AlthoughSarcinabacteria are often linked to lethal diseases, our results indicate thatSarcinaspp. appear to be a common member of the gut microbiota and seem to be an opportunistic pathogen. Further characterization and pathogenic analyses are required.