Temperature fields provide a noninvasive approach for manipulating individual macromolecules in solution. Utilizing thermophoresis and other secondary effects resulting from the inhomogeneous distribution of crowding agents, one may gain valuable insights into the interactions of molecular mixtures. In this report, we examine the steady-state concentration distribution and dynamics of DNA molecules in a poly­(ethylene glycol) (PEG)/water solution when exposed to localized temperature gradients generated by optical heating of a thin chrome layer at a liquid–solid boundary. This allowed us to experimentally investigate the interplay between DNA thermophoresis and PEG-induced entropic depletion effects. Our quantitative analysis demonstrates that the depletion effects dominate over DNA thermophoresis, causing the DNA polymers to migrate toward the heat source. Additionally, we explore the transient stretching of individual DNA molecules in thermally induced PEG gradients and estimate the contributing forces.