Transposable elements (TEs) are the most active, diverse and ancient component in a broad range of genomes. As such, a complete understanding of genome function and evolution cannot be achieved without a thorough understanding of TE impact and biology. However, in-depth analyses of TEs still represent a challenge due to the repetitive nature of these genomic entities. In this work, we present a broadly applicable and flexible tool: T-lex2. T-lex2 is the only available software that allows routine, automatic, and accurate genotyping of individual TE insertions and estimation of their population frequencies both using individual strain and pooled next-generation sequencing (NGS) data. Furthermore, T-lex2 also assesses the quality of the calls allowing the identification of miss-annotated TEs and providing the necessary information to re-annotate them. Although we tested the fidelity of T-lex2 using the high quality Drosophila melanogaster genome, the flexible and customizable design of T-lex2 allows running it in any genome and for any type of TE insertion. Overall, T-lex2 represents a significant improvement in our ability to analyze the contribution of TEs to genome function and evolution as well as learning about the biology of TEs. T-lex2 is freely available online at http://petrov.stanford.edu/cgi-bin/Tlex.html.