Morphologic overlap between renal oncocytoma (RO) and chromophobe renal cell carcinoma (ChRCC) can pose diagnostic challenges, especially in biopsy samples in which tumor quantity may be limited, architectural features are not well represented, and gross examination is not possible. It has been demonstrated that immunohistochemistry (IHC) for HNF1α, HNF1β, and S100A1 are differentially expressed in RO and ChRCC in resection specimens. We evaluated the utility of these markers in FNA and core biopsies of RO and ChRCC. IHC for HNF1α, HNF1β, and S100A1 were performed on 61 RO specimens (36 FNA biopsies and 25 core biopsies) and on 14 ChRCC specimens (10 FNA biopsies and 4 core biopsies), and results were scored semiquantitatively for staining intensity (0-3: negative, weak, moderate, strong) and staining extent (0-4: 0%, 1-25%, 26-50%, 51-75%, 76-100%). Forty-four (44) of 60 (73%) RO displayed moderate-to-strong nuclear reactivity for HNF1β compared to 3 of 14 (21%) ChRCC (P < .001). Staining was present in >50% of tumor cells in 34 of 60 (57%) RO and in 2 of 14 (14%) ChRCC (P = .004). S100A1 was moderately-to-strongly positive in 45 of 56 (80%) RO and in 1 of 13 (8%) ChRCC (P < .001), with 39 of 56 (70%) and 2 of 13 (15%) cases, respectively showing positivity in >50% of tumor cells (P< .001). No ChRCCs were positive for both markers. There was no statistically significant difference in intensity or extent of HNF1α staining between RO and ChRCC. HNF1β and S100A1 positivity was observed in a significantly greater proportion of RO than ChRCC. IHC for both markers can thus aid in the differential diagnosis. Accurate distinction is of increasing importance as tumor-ablative procedures and active surveillance become more widely adopted.