Pythium oligandrum is a parasite of cultivated Agaricus bisporus. Infection results in significant yield reductions and a disease referred to as ‘black compost’. In this study, P. oligandrum isolates were isolated from New Zealand mushroom composts, and their ribosomal DNA internal transcribed spacer (ITS) regions were amplified using the polymerase chain reaction (PCR). ITS nucleotide sequences obtained from New Zealand P. oligandrum isolates were compared with those previously identified P. oligandrum isolates and 23 described Pythium species. Although New Zealand P. oligandrum isolates had high ITS nucleotide identity with internationally identified P. oligandrum, the order of nucleotides in some regions varied when compared with other Pythium species. These varied nucleotides within the ITS region were used to design PCR primers (P.OLIG.F1 and P.OLIG.R04) for the specific amplification of a 384-bp fragment from P. oligandrum DNA. P.OLIG.F1 and P.OLIG.R04 were used to identify a major source of P. oligandrum inoculation on a New Zealand mushroom farm. Application of this diagnostic test will assist farming strategies implemented to prevent future P. oligandrum outbreaks. Furthermore, results presented identify a need for species resolution between P. oligandrum and P. hydnosporum.