In March 2024, highly pathogenic avian influenza A(H5N1) virus, clade 2.3.4.4b, was detected in dairy cows in the United States, and at the same time in resident cats on affected farms. To help guide sample collection and diagnosis in cats, here we report the distribution of lesions and detection of H5N1 clade 2.3.4.4b influenza A virus (IAV) infection by PCR, immunohistochemistry (IHC), and serology in samples from 4 deceased and 2 living cats from 3 separate affected dairy farms. Although gross lesions were not diagnostic, histologically, all 4 deceased cats had nonsuppurative and necrotizing encephalitis and subtle interstitial pneumonia, and some also had significant myocarditis (3 of 4), chorioretinitis (2 of 4), and sialadenitis (1 of 2). The virus was detected by IHC in the aforementioned tissues, and by PCR in each brain (Ct = 9.9-25.1), lung (17.4-32.7), oropharyngeal swab (28.3-30.5), urine (30.3-34.4), and nasal swab (33.5-34.1) collected postmortem; fecal swabs were PCR-negative. In the antemortem samples, the virus was detected by PCR in the oropharyngeal swabs (34.1-36.1), whole-blood samples (30.8-36.6), and one serum sample (31.7). Seroconversion was detected in one cat. Our results support histologic evaluation of brain, lung, eyes, and heart, and PCR testing of brain and lung for postmortem diagnosis, and show that oropharyngeal swabs, urine, serum, and whole blood are suitable samples for antemortem detection of IAV infection in clinically affected cats.