Autonomous non-long terminal repeat retrotransposons (NLRs) are ubiquitous mobile genetic elements that insert their DNA copies at new locations by retrotransposition. In vertebrates, there are 4 NLR clades, L1, L2, CR1, and RTE, which diverged in the Precambrian era. It has been demonstrated that retrotransposition of L1 and L2 members proceeds via coordinated reactions of targeted DNA cleavage and reverse transcription catalyzed by the NLR-encoded proteins, which are followed by the joining of the 5' (upstream) junction. However, the study on the mobility pathways for vertebrate NLRs is so far limited to L1 and L2. In this report, using target analysis of nested transposons for genomic copies, we studied retrotransposition pathways for a variety of vertebrate NLRs, including those of the L1, L2, CR1, and RTE clades in the human, cow, opossum, chicken, and zebrafish genomes. Thus, this study constitutes the first comprehensive analysis of NLR retrotransposition products in vertebrates. Our data revealed that these elements share similar mechanisms for the cleavages of the 2 target DNA strands and for the initiation of reverse transcription. Possible endonuclease-independent insertions were also identified. Overall, our results suggest the existence of multiple retrotransposition pathways that are conserved among the diverse NLR clades in various vertebrate hosts.