Small heat shock proteins (sHSPs) and related α-crystallins are virtually ubiquitous, ATP-independent molecular chaperones linked to protein misfolding diseases. They comprise a conserved core α-crystallin domain (ACD) flanked by an evolutionarily variable N-terminal domain (NTD) and semi-conserved C-terminal extension/domain (CTD). They are capable of binding up to an equal mass of unfolding protein, forming large, heterogeneous sHSP-substrate complexes that coordinate with ATP-dependent chaperones for refolding. To derive common features of sHSP-substrate recognition, I compared the chaperone activity and specific sHSP-substrate interaction sites for three different sHSPs from Arabidopsis (At17.6B), pea (Ps18.1) and wheat (Ta16.9), for which the atomic solution-state structures were modeled. I used the homobifunctional, amine- reactive, 7 Å cross-linker bis(sulfosuccinimidyl)glutarate (BS2G) to report on interaction sites between the sHSPs, a model substrate, malate dehydrogenase (MDH), and interactions between At17.6B and an endogenous target, fructose-1,6-bisphosphatealdolase (FBA). Interaction sites were identified by examining the position of residues that were modified by BS2G and coordinated cross-links with residues in other peptides, which were detected using ESI-LC-MS/MS. MDH exhibited major molecular rearrangement upon heat denaturation and association with sHSPs evidenced by detection of new MDH-MDH cross-links incompatible with the native structure. The NTD of all three sHSPs was a major site of cross-linking to substrate and was found linked to multiple sites on MDH, consistent with heterogeneous molecular interactions. While the N-terminus of Ta16.9 and At17.6B participated in many sHSP-MDH interactions, the more efficient Ps18.1 cross- linked MDH at multiple sites using all three sHSP domains. Not all sHSP or MDH Lys residues formed cross-links, despite displaying reactivity with the cross-linker, demonstrating preferred sites of interaction. At17.6B-FBA interactions closely resembled sHSP-model substrate data acquired with MDH, validating the cross-linking method and interaction models. Both BS2G and the UV-inducible, site specific cross-linker benzoylphenylalanine (Bpa), were used to detect direct sHSP-Hsp70 interactions during the substrate refolding process, suggesting that inter-chaperone interaction may play a role in the mechanismrequired for substrate handoff from the sHSP-substrate complex to Hsp70 for refolding.