The oleaginous yeast Yarrowia lipolytica is an important platform organism for biotechnology applications. In this study, we established an in vivo DNA assembly system leveraging CRISPR-Cas9 for efficient genomic integration of multiple DNA fragments into the genome of Y. lipolytica. Using the green fluorescent protein mNeonGreen as a model, we demonstrated 53% correct assembly of three DNA fragments with homology arms as short as 50 bp. The system was further validated by constructing 2-3 step biosynthetic pathways for pigments betaxanthin and betanin. To improve the homologous recombination efficiency of Y. lipolytica, we expressed S. cerevisiae RAD52 (ScRAD52) or a Cas9-hBrex27 fusion protein. While ScRAD52 expression impaired growth, the cas9-hBrex27 fusion enhanced integration efficiency, particularly for multifragment pathway assemblies. The in vivo assembly method simplifies pathway construction and gene overexpression in Y. lipolytica.