Over the years, pulsed-field gel electrophoresis (PFGE) has been proven a robust technique to type isolates with a high resolution and a good reproducibility. In this study, a PFGE protocol is described for the typing of Mycoplasma hyopneumoniae isolates. The potential of this technique was demonstrated by comparing M. hyopneumoniae isolates obtained from the same as well as from different herds. The use of two different restriction enzymes, SalI and ApaI, was evaluated. For each enzyme, the resulting restriction profiles were clustered using the unweighted pair group method with arithmetic means (UPGMA). For both obtained dendrograms, the included isolates of the related M. flocculare species clustered separately from all M. hyopneumoniae isolates, forming the root of the dendrograms. The PFGE patterns of the M. hyopneumoniae isolates of different herds were highly diverse and clustered differently in both dendrograms, illustrated by a Pearson's correlation coefficient of only 0.33. A much higher similarity was observed with isolates originating from different pigs of a same herd. The PFGE patterns of these isolates always clustered according to their herd and this for both dendrograms. In conclusion, the results indicate a closer relationship of M. hyopneumoniae isolates within a herd compared to isolates from different herds and this for both restriction enzymes used. Since the described PFGE technique was shown to be highly discriminative and reproducible, it will be a helpful tool to further elucidate the epidemiology of M. hyopneumoniae.