Clostridium perfringens, a commensal of the intestinal tract of many animal species, has been associated with necrotic enteritis (NE), an economically significant poultry disease. Clostridium perfringens is known to survive in the environment for extended periods of time through the formation of spores. These spores have the potential to be transmitted to subsequent flocks. Persistence of a single C. perfringens strain in a broiler chicken farm environment has, however, been poorly documented. The aim of this study was to compare multiple isolates of C. perfringens collected over time in a single farm with recurrent episodes of NE. Isolates were recovered from the intestines of chickens affected with NE (2014 and 2016 outbreaks) and from healthy chickens (2017), as well as from environmental samples (2016). PCR characterization of those isolates showed that all sampling groups contained netB-positive isolates except for the environmental samples. Moreover, results showed that all environmental isolates were positive for the cna adhesin whereas other groups had lower numbers of cna-positive isolates. Biofilm formation assays showed that most of the isolates were able to form biofilm. Pulsed-field gel electrophoresis analysis showed that one clone was present in every sampling group, with the exception of the 2014 outbreak. However, one clone found in the latter group was highly similar, having 94% similarity with the persistent C. perfringens clone. This study describes for the first time the persistence of a C. perfringens strain on a broiler chicken house over a 3-yr period.