Introduction Pulmonary invasive mold infections (IMI) remain a significant cause of morbidity and mortality after hematopoietic cell transplant (HCT). Non-invasive diagnostic options are limited, particularly for non-Aspergillus molds. Given differences in spectrum of activity and toxicities associated with mold-active therapy, early diagnosis and targeting of specific IMI is important. Objectives We evaluated the performance of the Karius cell-free plasma next-generation sequencing (NGS) test for detecting IMI in HCT recipients with pneumonia. Methods We conducted a retrospective case-control study of 57 HCT recipients with proven/probable pulmonary IMI (6 were non-Aspergillus) based on the 2008 EORTC criteria and 19 controls with non-fungal pulmonary infections. All subjects had frozen plasma samples obtained within 14 days of diagnosis (76% within 7 days). Diagnostic workup included computed tomography chest imaging and bronchoalveolar lavage (BAL) and/or biopsy. Fungal stains and culture were performed on BAL fluid and tissue samples, and galactomannan testing was performed on BAL and serum. Cell-free DNA was extracted from plasma and NGS performed (Karius, Redwood City, CA). Human reads were removed and remaining sequences aligned to a curated database including over 300 fungi. Organisms present above a predefined significance threshold were reported. Analysis of sequencing data was blinded to all clinical data. Results Among 57 proven and probable cases of IMI, we identified pathogenic molds in 15 subjects (26%). Among the subset of 44 subjects with a plasma sample +/- 7 days from diagnosis, the rate of detection increased to 32% (14/44). Mold was not identified in any controls. In non-Aspergillus proven IMI, the Karius test identified 83% (5/6) of molds. In contrast, in Aspergillus proven/probable disease, Aspergillus fumigatus was identified in 13.7% (7/51) of subjects. All Aspergillus positives had a plasma sample available +/- 7 days from diagnosis. In three other subjects with Aspergillus proven/probable disease, we identified Rhizomucor miehei, Rhizomucor pusillus, and Cunninghamella, which could be consistent with the clinical findings. By optimizing the analysis pipeline for sensitivity, an additional 6 subjects with Aspergillus spp and other pathogenic molds were identified, increasing the detection rate of pathogenic molds to 31.4% (16/51). Conclusion The Karius cell-free plasma NGS test is a non-invasive means of detecting IMI, particularly non-Aspergillus molds in HCT recipients with pulmonary disease. This may be a useful adjunctive test for diagnosing IMI in specific clinical contexts, and larger studies should be conducted.