The difficulty in correctly identifying the etiologic factors of premature ejaculation (PE) could be due to the fact that the role of the pelvic floor muscles (PFMs) in the voluntary control of ejaculatory reflex has not been elucidated. The aim of the present investigation was to measure the prevalence of awareness of the role and use of PFM contraction in controlling the ejaculatory reflex among PE and non-PE participants. A total of 44 men with PE and 73 men without PE were recruited. In the first part of the study, we validated a test that rendered the participants aware of the PFMs through digital rectal examination and the PFM contraction. In the second part, we posed this multiple-choice question: "Which muscles do you use to delay ejaculation?". Men not answering correctly were considered not to be using the PFMs and also to be unaware that it is necessary to contract the PFMs to control the ejaculatory reflex. Only 3 of 44 subjects (6.8%) with PE and 60 of 73 subjects (82%) without PE answered correctly and used PFMs to control the ejaculatory reflex (Fisher test p < 0.0001). This test has a sensibility of 93%, a specificity of 82%, and an accuracy of 86%. The vast majority of PE subjects were unaware that to inhibit or delay ejaculation it is necessary to contract the PFMs. This association also raises the question whether the difficulties in defining PE and finding effective PE therapies could be due to a nonhomogeneous population of PE patients with different etiopathogenetic factors. More studies are required to confirm these data and to answer this question.