<p>Myofibroblasts were recently identified as the most critical cells responsible for epiretinal membrane contraction, however the myofibroblast number in different types of membranes may differ substantially. In this retrospective study, we compare myofibroblast distribution and density in idiopathic epiretinal membranes (iERMs) and epiretinal membranes (ERMs) in proliferative diabetic retinopathy (PDR). Samples (<i>n</i> = 42) from iERM and from thin ERM, ERM, and epiretinal fibrovascular membrane (FVM) in PDR (thin ERM/PDR, ERM/PDR, and FVM/PDR, respectively) were collected via a standard 27-gauge vitrectomy. They were analyzed using flat-mount immunohistochemical staining with α-smooth muscle actin. Cell densities (in cells/mm²) for iERM, thin ERM/PDR, ERM/PDR, and FVM/PDR were 339.00 ± 256.76, 12.39 ± 7.46, 114.22 ± 40.51, and 844.15 ± 323.21, respectively, and significantly different (<i>P</i> &lt; 0.001). Myofibroblast densities (in cells/mm²) in these groups were 39.48 ± 46.51, 0.65 ± 0.95, 9.22 ± 12.08, and 303.43 ± 57.21, respectively, and significantly different (<i>P</i> &lt; 0.001). Myofibroblast density in iERM was higher than that of thin ERM/PDR (<i>P</i> &lt; 0.001), higher but not significantly different from ERM/PDR (<i>P</i> = 0.077), and lower than that of FVM/PDR (<i>P</i> &lt; 0.001). The myofibroblasts/total cell ratios in these groups were 12.66% ± 12.38%, 5.89% ± 9.78%, 9.55% ± 10.86%, and 40.35% ± 14.37%, respectively, and significantly different (<i>P</i> &lt; 0.001). Myofibroblast density was strongly correlated with cell density and the myofibroblast/total cell ratio (<i>P</i> &lt; 0.001).</p>