Molecular cloning is a cornerstone of biomedical, biotechnological, and synthetic biology research. As such, improved cloning methodologies can significantly advance the speed and cost of research projects. Whereas current popular cloning approaches use in vitro assembly of DNA fragments, in vivo cloning offers potential for greater simplification. It is generally assumed that bacterial in vivo cloning requires Escherichia coli strains with enhanced recombination ability; however, this is incorrect. A widely present, bacterial RecA-independent recombination pathway is re-emerging as a powerful tool for molecular cloning and DNA assembly. This poorly understood pathway offers optimal cloning properties (i.e. seamless, directional, and sequence-independent) without requiring in vitro DNA assembly or specialized bacteria, therefore vastly simplifying cloning procedures. Although the use of this pathway to perform DNA assembly was first reported over 25 years ago, it failed to gain popularity, possibly due to both technical and circumstantial reasons. Technical limitations have now been overcome, and recent reports have demonstrated its versatility for DNA manipulation. Here, we summarize the historical trajectory of this approach and collate recent reports to provide a roadmap for its optimal use. Given the simplified protocols and minimal requirements, cloning using in vivo DNA assembly in E. coli has the potential to become widely employed across the molecular biology community.