Penicillium marneffei is the most important thermal dimorphic pathogenic fungus in Southeast Asia. Despite findings on diverse genes and mechanisms involved in dimorphic switching, the key to signally pathways governing the switch is still unknown. Since miRNAs are important regulatory molecules in eukaryotes, we attempt to define if miRNAs are expressed in different growth phases of P. marneffei. Using high-throughput sequencing, we identified 24 potential milRNA candidates in P. marneffei, which were more abundantly expressed in mycelial than yeast phase. Two genes, dcl-1 and dcl-2, encoding Dicer-like proteins and the gene, qde-2, encoding Argonaute-like protein, were also identified. Phylogenetic analysis showed that dcl-2 of P. marneffei was more closely related to the homologues in other thermal dimorphic pathogenic fungi than to Penicillium chrysogenum and Aspergillus spp. dcl-2 demonstrated higher mRNA levels in mycelial than yeast phase. Northern blot analysis confirmed expression of two milRNAs, PM-milR-M1 and PM-milR-M2, only in mycelial phase, whose expression was dependent on dcl-2 but not dcl-1 or qde-2. The mRNA levels of three predicted targets of PM-milR-M1 were upregulated in knockdown strain PM-milR-M1KD, supporting its regulatory function. This study represents the first discovery of milRNAs in thermal dimorphic fungi, with differential expression in different growth phases.