The extent to which demographic characteristics of educational heads differentiate their instructional leadership practices has been a contentious matter for several decades. The aim of this study was to investigate the instructional leadership practices of school heads as well as examine the extent to which headteachers’ gender, professional qualification, and work experience differentiated their instructional leadership practices in public basic schools in Ghana. This was a quantitative study underpinned by the positivist paradigm. The study employed the descriptive survey research design where 127 headteachers and 643 teachers were selected through census and proportionate stratified random sampling techniques respectively. The Principal Instructional Management Rating Scale (PIMRS) was adapted from Hallinger and Murphys (1985) and used to collect numerical data for the study which was analysed using descriptive statistics like mean and standard deviation as well as inferential statistics including independent samples t-test and one-way analysis of variance (ANOVA). The findings revealed that defining school mission dimension of instructional leadership practice was most prevalent among the headteachers as compared to managing the instructional programme and protecting school climate respectively. Additionally, the findings established that gender accounted for differences among headteachers in promoting a positive school climate where male headteachers rated higher than their female peers. Furthermore, the study established that work experience of the headteachers led to significant differences in defining school mission, managing instructional programme, promoting a positive school climate as well as the overall instructional leadership practice. Therefore, the study concluded that headteachers’ gender matters in promoting a positive climate in the schools while the work experience of the headteachers is a determinant of differences in their instructional leadership practices in the schools. Based on these findings, the study recommended that Ghana Education Service through the Regional and District Directorates of Education should liaise with instructional leadership specialists to organise in-service and refresher training programmes for headteachers on instructional leadership practices so as to sustain, improve, and intensify its practice in public basic schools.