Layer 1 (L1) interneurons (INs) play a key role in modulating the integration of inputs to pyramidal neurons (PNs) and controlling cortical network activity. Hyperpolarization-activated, cyclic nucleotide-gated, non-specific cation (HCN) channels are known to alter the intrinsic and synaptic excitability of principal components (PCs) as well as select populations of GABAergic INs. However, the developmental profile and functional role of HCN channels in diverse L1 IN populations is not completely understood. In the present study, we used electrophysiological characterization, in conjunction with unbiased hierarchical cluster analysis, to examine developmental modulation of L1 INs by HCN channels in the rat medial agranular cortex (AGm). We identified three physiologically discrete IN populations which were classified as regular spiking (RS), burst accommodating (BA) and non-accommodating (NA). A distinct developmental pattern of excitability modulation by HCN channels was observed for each group. RS and NA cells displayed distinct morphologies with modulation of EPSPs increasing in RS cells and decreasing in NA cells across development. The results indicate a possible role of HCN channels in the formation and maintenance of cortical circuits through alteration of the excitability of distinct AGm L1 INs.