Factors influencing Clostridium botulinum contamination in the honey production environment were evaluated in a 3-year survey. A number of 1,168 samples from 100 apiaries and related facilities were analysed for the presence of C. botulinum types A, B, E and F, using multiplex polymerase chain reaction targeted to botA, botB, botE and botF genes. Production methods and environmental factors were registered using a questionnaire and by personal observation. Clostridium botulinum was shown to be a common finding throughout the whole honey production chain, and the type most frequently detected was group I type B. In a pulsed-field gel electrophoresis (PFGE) analysis of 202 group I type B isolates, only six different PFGE profiles were observed, of which two clearly distinct profiles predominated. This may indicate the existence of at least two different genetic lineages. The high prevalence of C. botulinum in soil and in samples associated with beeswax suggests the accumulation of soil-derived botulinal spores in wax. Additionally, according to Spearman's rank order correlation and multivariate analysis, production hygiene-dependent factors have a significant influence on the contamination, and thus the number and frequency of C. botulinum spores in honey could possibly be diminished by increasing hygienic level in honey production.