The aim of the present study was to analyse the genetic diversity of the alpha-toxin encoding plc gene and the variation in alpha-toxin production of Clostridium perfringens type A strains isolated from presumably healthy chickens and chickens suffering from either necrotic enteritis (NE) or cholangio-hepatitis. The alpha-toxin encoding plc genes from 60 different pulsed-field gel electrophoresis (PFGE) types (strains) of C. perfringens were sequenced and translated in silico to amino acid sequences and the alpha-toxin production was investigated in batch cultures of 45 of the strains using an enzyme-linked immunosorbent assay (ELISA) approach. Overall, the truncated amino acid sequences showed close similarity (>98% at the amino acid level) to previously reported sequences from chicken-derived C. perfringens isolates. Variations were however observed in 23 out of 379 aa positions leading to the definition of 26 different alpha-toxin sequence types among the 60 strains. Moreover, a type II intron of 834 non-coding nucleotides was identified in the plc gene of three of the investigated strains. The in vitro alpha-toxin production investigated in 45 of the strains, including the three harbouring the intron, revealed no correlation between PFGE type, alpha-toxin sequence type, health status of the host chickens and level of alpha-toxin production. It is therefore concluded that neither plc gene type nor alpha-toxin production level seems to correlate to origin (healthy or diseased chicken) of the C. perfringens strains.