Background: There have been criticisms that classroom teachers are insufficiently trained and lack the confidence to deliver high-quality physical education (PE). Physical literacy (PL) is one of the main concepts that encompass various outcomes and is the main focus of the PE curriculum in many countries. However, to be able to transfer this variety of outcomes encompassed by PL to students, it is necessary for teachers to have some qualities and theoretical comprehension of the philosophy of PL. Teacher training programs and the experiences of teachers provide a base for the transfer of the necessary PL and PE qualities to teachers. However, in Turkey, the training programs for classroom teachers do not include sufficient PE courses. Purpose: To explore the differences between PE and classroom teachers in respect of PL according to their experience level and gender. Methods: The study sample included 161 PE teachers working in middle and high schools and 124 classroom teachers working in primary schools across Turkey. The Turkish version of the 'Perceived Physical Literacy Scale for Physical Education Teachers'] was used to collect PL data. This scale is comprised of the 3 factors of 'knowledge and understanding', 'sense of self and self-confidence', and 'communication'. Data were analyzed using 3- way Multivariate Analysis of Variance (3-way MANOVA). Results: The 3-way MANOVA results indicated a significant main effect on the PL of teacher category and teaching experience (p < 0.01). Results also indicated a significant interaction effect of teacher category and teaching experience, and gender and teaching experience (p < 0.05). Post-hoc test results indicated that PE teachers had significantly higher mean scores than classroom teachers and teachers with 16--20 years of experience had significantly lower mean scores in the 'sense of self and self-confidence' and 'communication' subscales, (p < 0.05). Pairwise comparisons indicated that these differences arose from differences between the classroom teachers. Discussion: The difference between PE and classroom teachers could be attributed to content differences in teacher training programs. The differences between those with different levels of experience were most probably associated with the reforms of classroom teacher training programs. The teachers with the lowest mean PL scores were classroom teachers with 16-20 years of experience, who had first structured PE courses during their pre-service training. Thus, the way that PE content is implemented in education faculties is important for the transfer of critical knowledge to prospective teachers. Conclusion: The PE content in classroom teacher training programs is insufficient for the development of an awareness of the importance of PL for child development. Supporting classroom teachers with in-service training is recommended to improve both the comprehension of PL and the quality of PE. These results demonstrate the importance of additional PE content in classroom teacher training programs as well as how this content is delivered.