We used site-directed mutagenesis and labeling of the type 1 ryanodine receptor (RyR1) recombinantly expressed in HEK-293T cells to identify RyR1 residues that interact with FKBP12, a constitutively-bound protein that promotes the closed state of the RyR1 calcium channel. While the location of bound FKBP on the RyR1 cryo electron microscopic (EM) map is known, the RyR1 sequence elements forming the FKBP binding site are poorly understood. Previously, we have shown that a His10 tag inserted at RyR1 position 620, adjacent to the N-terminal “ABC” domains, abolished FKBP binding. Here, we used structural modeling to predict loop regions in downstream domains, and then used these loops for His10 insertion sites. Fluorescent FKBP (F-FKBP) binding was then assessed in functional, full-length RyR1 containing these His10 tags. We determined that insertion of His10 tags in specific loops could completely abolish F-FKBP binding, even though RyR1 calcium release activity was unaffected. Scrambling the amino acid sequence of the loops identified using His10 tags also eliminated F-FKBP binding. The effects of disrupting these loops on F-FKBP binding were far more pronounced compared to previously established FKBP binding motifs, such as the VP dipeptide at RyR1 position 2461 and the PKA phosphorylation site at position 2843 within the PKA/CaMKII phosphorylation loop of RyR1. Thus, our results identify novel major FKBP binding determinants in RyR1. Supported by NIH and Canadian grants R01AR059126 (to JDF) R01HL092097 (to RLC), and CIHR 259009 (to FVP).