Neuropeptide Y (NPY) is an abundant neuropeptide of the neocortex involved in numerous physiological and pathological processes. Due to the large electrophysiological, molecular and morphological diversity of NPY-expressing neurons their precise identity remains unclear. To define distinct populations of NPY neurons we characterized, in acute slices of rat barrel cortex, 200 cortical neurons of layers I-IV by means of whole-cell patch-clamp recordings, biocytin labeling and single cell Reversed Transcriptase-Polymerase Chain Reaction (scRT-PCR) designed to probe for the expression of well established molecular markers for cortical neurons. To classify reliably cortical NPY neurons we used and compared different unsupervised clustering algorithms based on laminar location, electrophysiological and molecular properties. These classification schemes confirmed that NPY neurons are nearly exclusively gamma-aminobutyric acid (GABA)-ergic and consistently disclosed three main types of NPY-expressing interneurons. (1) Neurogliaform-like neurons exhibiting a dense axonal arbor, were the most frequent, superficial, and substantially expressed the neuronal isoform of nitric oxide synthase (NOS-I). (2) Martinotti-like cells characterized by an ascending axon ramifying in layer I co-expressed somatostatin (SOM) and were the most excitable type. (3) Among fast spiking (FS) and parvalbumin (PV) positive basket cells, NPY expression was correlated with pronounced spike latency. By clarifying the diversity of cortical NPY neurons, this study establishes a basis for future investigations aiming at elucidating their physiological roles.