The erosion of demethylation inhibitor (DMI) fungicides effectiveness over time in Europe has been attributed to mutation sites in the 14α-demethylase encoded by the nuclear gene CYP51 (Cools and Fraaije 2013). These mutations first appeared in Europe in the 1990s, became widespread over the next 20 years, and were also recently reported in North America (Estep et al. 2015; Lucas et al. 2015). Zymoseptoria tritici has been present and causing disease on wheat (Triticum aestivum) in Australia for many decades and the use of fungicides for its control has become more common over the past 15 years. However, no significant changes in the field performance of fungicides have been noted by growers. To investigate possible undetected changes, we sequenced the CYP51 gene of 18 isolates cultured from wheat leaves collected in a commercial field on 1 July 2012 at Inverleigh, Victoria (–38.086743° S; 143.935783° E), and 3 isolates cultured from wheat leaves collected from trial plots on 22 August 2002 at Wagga Wagga, NSW (–35.044544° S; 147.316318° E). The nucleotide sequence of Z. tritici strain ST1 eburicol 14 alpha-demethylase (CYP51) gene (GenBank Accession No. AY730587.1) was used to design a set of three forward and three reverse sequencing primers across the known mutations. The sequence reads were assembled into contigs and checked for complete sequence. To generate an alignment of mature protein sequences, the mature protein sequence of ST1 (CYP51) gene was downloaded from NCBI to use as a reference. CYP51 nucleotide sequences were translated into amino acid sequences using all six possible frame shifts. These were aligned to the mature protein sequence of ST1 (CYP51) to identify the correct frame-shift sequence, enable the removal of intron sequences, and finally generate an amino acid alignment. Nucleotide and amino acid sequences are deposited in GenBank with Accession Nos. KT201543 to KT201563. Sixteen of the isolates from Victoria were carrying the Y137F mutation, one isolate carried the L50S-Y461S mutation, and one the L50S-S188N-N513K. The three isolates from NSW collected in 2002 contained no mutations compared with the reference accession. Fungicide sensitivities for propiconazole were determined at 50% effective concentrations (EC50) using rates of 100, 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003, and 0.001 mg/liter, and the resistance factor (RF) of each isolate was calculated as fold change in the EC50 compared with that of the wild-type strains as per Cools et al. (2011), four replicates per isolate were performed. Results of the phenotypic assay of a subset of nine isolates (four Y137F mutants, one L50S-Y461S, one L50S-S188N-N513K, and three Wild-type) confirmed the elevated EC50 values for the mutations known to cause reduced sensitivity. The Y137F and L50S-Y461S mutants showed EC50 values of 1.08 and 1.03 mg/liter and RF levels in the range of 3.07 and 2.93. respectively, while the wild-type and L50S-S188N-N513K mutant had EC50 of 0.352 and 0.367 mg/liter, respectively. The EC50 values observed for the isolates appear higher than those found by Cools et al. (2011); however, the RF values are lower for both mutations. Further exploration of CYP51 mutations occurring in Z. tritici within the Australian cropping regions will be necessary to establish how widespread the Y137F and L50S-Y461S mutants are, and if others such as the S524T or the V136A, which are associated with higher levels of resistance to DMIs, are also present.