Gelation, the irreversible loss of fluidity in freeze-thawed hen egg yolk, is undesirable to food processors because of reduced yolk dispersibility and functionality. The mechanism of gelation has not yet been fully elucidated. The objective of this research was to determine the gelation mechanism by studying the gel mechanical properties, particle size distribution, protein characteristics, free water content, matrix mobility, and microstructural arrangement of fresh vs gelled yolk in a long-term frozen storage study. A separate investigation was conducted for the development of a sensitive and robust proton nuclear magnetic resonance (1H NMR) spectroscopic technique to study matrix mobility in gelled yolk. The detected yolk components were successfully identified as protein, lipid, and water after optimization of operating conditions, and results showed good repeatability. The long-term study of yolks stored at -20°C for varying lengths of time (1, 3, 7, 14, 28, 84, 168 d) before thawing showed that both plasma and granules of yolk contributed to gelation. Gel strength and particle size increased with increasing storage time, indicating aggregation of yolk constituents. Polyacrylamide gel electrophoresis (PAGE) results suggested the aggregation of proteins, particularly phosvitin of the granules. Intermolecular disulfide bonding between high-density lipoproteins may also have occurred. Two stages of gelation were observed with particle size analysis and 1H NMR spectroscopy. Lipoproteins or apolipoproteins may have aggregated in the first stage (≤ 84 d) of gelation. In the second stage (≥ 84 d), proteins or apolipoproteins may have aggregated after dissociating from aggregates formed during the first stage. The dissociation of granular components during long-term freezing was also observed via transmission electron microscopy (TEM). Disruption of the freeze-thawed yolk matrix confirmed that ice crystal growth was required for gelation to occur, though no obvious LDL aggregation was observed.