An increased number of cases of salmonellosis caused by the rare serotype Mikawasima was noted in the years 2012 and 2013 in the Czech Republic. A potential on-going outbreak caused by this serotype in European countries has also been reported. Altogether 14 human and 1 environmental isolates from the year 2012 and 11 human isolates from the year 2013 from different locations of the Czech Republic were sent to our laboratory for typing. Macrorestriction analysis together with antimicrobial susceptibility testing and PCR for ESBL and plasmid-mediated quinolone resistance detection were performed to compare our isolates. Twenty-one isolates created two very similar clusters and 5 isolates had different profiles. Twenty-five isolates were fully susceptible to all agents used. One isolate showed resistance to 12 microbial agents and possessed blaTEM, blaSHV, blaOXA, blaCTX-M and qnrB genes.