Northern Spotted Owls (Strix occidentalis caurina) are of management and conservation concern in the US Pacific Northwest where populations have been monitored since the 1990s using mark-resight methods. Passive acoustic monitoring has the potential to support monitoring efforts; however, its use is currently primarily restricted to determining species presence rather than breeding status. Distinguishing female from male Northern Spotted Owl vocalizations could facilitate determination of pair status using passive acoustic methods, greatly enhancing inference derived from noninvasive monitoring data. In 2017, we deployed 150 autonomous recording units (ARUs) within 30 5-km2 hexagons overlapping recently occupied owl territories in Oregon and Washington, USA, where mark-resight methods were simultaneously occurring. We collected approximately 150,000 hours of recordings and detected 22,458 Northern Spotted Owl vocalizations at 76 ARUs. We summarized vocalizations by call type and found differences in the proportion of call types made by single, paired, and nesting owls. We used expert opinion to classify 2812 four-note location calls as female or male. We summarized inter-sex variation within 19 acoustic attributes of the four-note location call and its subcomponents, and developed a mixed logistic regression model to classify sex based on call-segment acoustic attributes. Males generally called at lower frequencies than females, with mean fundamental frequencies of 556 Hz and 666 Hz, respectively. Male four-note location calls were also longer than female calls, with signal median times of 1.99 s and 1.71 s, respectively. The middle-two-note and the full-call segment of the four-note location call were both useful for classifying sex of the calling owl. Our top-ranked models were able to predict 82–83% of our testing data consistent with expert classification as either male or female with 98–99% accuracy (17–18% of test set was classified as unknown). Our results suggest that acoustic characteristics of Northern Spotted Owl calls captured with ARUs can be used to identify whether sites have males and/or females present, and we suggest that further investigation into the full repertoire of Northern Spotted Owl call types is warranted.