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This study aimed to assess the pedagogical content knowledge of new literacies (PCKNL) among preservice teachers in reputable teacher education programs in the United States. I developed an instrument—the PCKNL survey—that assessed preservice teachers’ pedagogical content knowledge of new literacies. The survey was administered to a sample of preservice teachers in different preparation programs. Using confirmatory factor analysis, the constructs of the instrument were tested and shown to be reliable and valid. It is expected that this instrument will be used in teacher preparation programs. One of the implications of this study is that exists a need to create literacy curriculum that reflects elements of new literacies.