Learning in blended environments has become a ubiquitous part of student experience in tertiary education worldwide. Although students’ perceptions of learning environments are a key element in the learning process, there is a dearth of valid instruments to assess students’ perceptions in blended contexts. This study described the initial development and validation of a Perceptions of the Blended Learning Environment Questionnaire (PBLEQ). The analyses, involving two cohorts of students enrolled in courses either from humanities/social sciences disciplines or from sciences/engineering disciplines, consistently supported the bifactor model over a correlated first-order model and a second-order model. The bifactor model had a single perceptions factor that underlined each of the items. Separately, there were three specific factors: the perceptions of integration between face-to-face and online learning, the perceptions of online contributions, and the perceptions of the online workload, each having its own separate set of items. The invariance tests among the two cohorts validated that the PBLEQ had invariant factor structure, factor loadings, and intercepts. The PBLEQ has potential to help unravel students’ perceptions of the blended learning environment in diverse academic disciplines.