Fatty liver (FL) is a common condition affecting dairy cows during the peripartum period, with no diagnostic test that is accurate, fast, and low cost. FL can be determined by TG extraction, histopathology or flotation of liver biopsies from live animals. However, these methods are invasive, and at higher risk of hemorrhages, infection and adhesions. Cytology for detection of FL in cattle has not been extensively evaluated. It is feasible in clinical practice, being minimally invasive and inexpensive, and having a rapid turnaround time compared with biopsy. The objective was to estimate the sensitivity (Se) and specificity (Sp) of needle aspiration cytology and histopathology for FL using TG concentrations as the gold standard. Liver samples were harvested from a slaughterhouse in WI, USA. Livers were visually inspected for gross evidence of fat accumulation and classified as follow. (1): Normal—homogeneous maroon color (n = 20), (2): Moderate—moderate yellow-pale color (n = 20) and (3): Severe—more extreme yellow color (n = 20). Biopsies and needle aspiration were carried out from the parietal upper portion of the caudate lobe. Two samples of liver tissue were harvested with a 16 g x 15 cm biopsy needle for histological and TG concentration assessment. A third sample was harvested for cytology using an 18 g × 3.81 cm needle. The content of the needle was transferred to a glass slide, spread and air-dried. TG were assessed by a kit based on colorimetry/fluorimetry. Concentrations <2% were considered normal. Histological and cytological evaluation was blind to the visual classification and conducted by 2 pathologists. Se, Sp, and area under the receiver-operating curve [ROC] were calculated. For cytology, Se and Sp were 73% (95% CI 55.9–86.2) and 85% (95% CI 62.1–96.8), respectively. The area under ROC was 0.79 (95% CI 0.68–0.89). For histopathology, Se and Sp were 45.9% (95% CI 29.5–63.1) and 100% (95% CI 83.2–100), respectively. The area under ROC was 0.73 (95% CI 0.64–0.81). This data suggests that cytology may be a viable less invasive tool for detecting FL in dairy cattle.