Locked nucleic acids (LNA) are modified nucleotides where a ribonucleoside is linked between the 2'-oxygen and the 4'-carbon atoms with a methylene unit. LNA oligonucleotides exhibit increased thermal stability toward complementary DNA and RNA with characteristically higher melting temperatures. In situ hybridization (ISH) using LNA oligonucleotide probes targeting fungal ribosomal RNA (rRNA) sequences has not been described. This study details an ISH procedure by using a biotin-labeled LNA probe targeting Aspergillus spp.18s rRNA sequences. A genus-specific 3' terminally biotin-labeled oligonucleotide probe was commercially synthesized by using a mixture of DNA (60%) and LNA (40%). A rapid, 2-hour, nonisotopic ISH procedure was developed and performed on 20 culturally proven formalin-fixed, paraffin-embedded (FFPE) cases of Aspergillus spp. By ISH, the LNA probe effectively detected Aspergillus spp. rRNA sequences in all specimens. Compared with a DNA probe with the same sequence, the LNA probe produced a stronger signal. ISH with the Aspergillus-specific LNA probe was negative on culture-proven cases of other fungal pathogens including Zygomyces and Fusarium. ISH with an LNA oligonucleotide probe targeting Aspergillus 18s rRNA sequences is useful for rapidly detecting Aspergillus spp. in paraffin-embedded tissue specimens. This test could be used when fungal pathogens are observed in tissue but cultures are negative or have not been performed. ISH with LNA probes may be useful for detecting a variety of fungal pathogens in formalin-fixed, paraffin-embedded tissue specimens.