Acute invasive fungal rhinosinusitis (AIFRS) most commonly occurs in immunosuppressed patients. The identification of fungal subtypes is important for management, and cultures can be negative. We studied 55 specimens from 23 patients with AIFRS (Rhizopus sp, 6; Aspergillus sp, 8; Fusarium sp, 1; Alternaria sp, 1; and culture negative, 7) using in situ hybridization (ISH) with biotin-labeled oligonucleotide probes targeting Aspergillus sp, Fusarium sp, Rhizopus sp, and a sequence identified in dematiaceous fungi. Ribosomal RNA preservation was established by using a pan-fungal probe. Nucleic acid preservation was seen in 18 patients (33 specimens [60%]). ISH using the specific fungal probes highlighted the respective fungal organisms in all culture-positive cases with adequate negative controls. Of the 7 culture-negative AIFRS cases, 4 had preserved fungal sequences. Of these cases, 2 additional cases of Aspergillus and 1 additional case of dematiaceous species were identified. In our study, 60% of AIFRS cases had fungal nucleic acid preservation. ISH can effectively identify fungi in AIFRS. ISH for specific fungal pathogens may aid in species identification in specimens with negative cultures.