In order to evaluate the genetic variability of the causative agent of cold water disease (CWD), plasmid profiling was used to characterize Flavobacterium (F.) psychrophilum isolates (n = 169). Size analysis of plasmids in F. psychrophilum isolates (n = 128) from several fish species demonstrated that six kinds of plasmids were harbored, and ayu isolates had different profiles compared to other isolates. Moreover, multiple isolates (n = 41) from CWD outbreaks in 2002 to 2003 at a single ayu farm were examined to determine differences between isolates from successive outbreaks and showed different profiles by the sources of seedlings.