DNA nanostructures are typically assembled using a thermal annealing protocol by heating the DNA mixture to high temperatures and then cooling it down to a lower temperature. Recent efforts have shown the assembly of DNA nanostructures by incubation at constant temperatures in a process called isothermal assembly. DNA motifs, polyhedra, lattices, and other nanostructures based on single-stranded tiles and the DNA origami strategy have all been constructed using the isothermal assembly process. Several additives such as denaturing agents, cationic amino acids, and natural products aid in the isothermal process at room temperature and physiological temperature. This review focusses on the developments in isothermal assembly of DNA nanostructures, key takeaways from recent studies, and the advantages and limitations of isothermal assembly in the broader context of DNA nanotechnology.