Aims: In order to improve the future reliability of surveillance for Clostridium difficile infection (CDI), an Australia/New Zealand-wide survey was conducted to examine methods of laboratory diagnosis in use, identify deficiencies in practice and burden of CDI.Methods: An online survey of 48 Australian and New Zealand microbiology laboratories (private and public) was conducted in late 2009 and 2010 to collect information about methods of detection in use and collective testing experience from July 2008 to June 2009.Results: The overall prevalence (proportion positive of all faecal specimens tested) of C. difficile in 123 574 tested samples was 5.3%. The incidence rate across jurisdictions varied between 18.0 per 100 000 population in Victoria to 35.8 per 100 000 population in Tasmania, with a mean for Australia of 25.6 per 100 000 population. The incidence rate in New Zealand was 21.5 per 100 000 population. Most laboratories (60%) screened stools with an enzyme immunoassay (EIA) or equivalent that detected both toxins A and B.Conclusions: The low overall rates reported here may reflect the lack of sensitivity of diagnostic testing procedures currently used in Australia and New Zealand to detect C. difficile. Recommendations for best practice in diagnosis of C. difficile were developed by the Australasian Society for Infectious Diseases (ASID) C. difficile working party and later endorsed by ASID.