Successive structural changes of bacteriophage λ upon heating were characterized with quantitative experimental methods. In the commonly used Tris-Mg buffer, differential scanning calorimetry measurements first established that the protein capsid of λ phage melts at 87 °C and its genomic DNA melts at 91 °C. Interestingly, prior to the capsid melting, λDNA was found to escape out of the capsid and subject to DNase digestion above ~68 °C, as concluded from light scattering, UV absorption, and electron microscopy studies. Further investigations indicated distinct temperature-dependent behaviors of the three phage proteins. Around 68 °C, disruption of the tail first occurs and leads to the escape of λ DNA; above the capsid melting temperature of 87 °C, the auxiliary protein gpD of the phage head remains soluble in solution and resists centrifugal sedimentation, whereas the major capsid protein gpE is easily precipitated and likely exists as aggregates.