Molecular recognition by synthetic peptides is growing in importance in the design of biosensing elements used in the detection and monitoring of a wide variety of hapten bioanlaytes. Conferring specificity via bioimmobilization and subsequent recovery and purification of such sensing elements are aided by the use of affinity tags. However, the tag and its site of placement can potentially compromise the hapten recognition capabilities of the peptide, necessitating a detailed experimental characterization and optimization of the tagged molecular recognition entity. The objective of this study was to assess the impact of site-specific tags on a native peptide's fold and hapten recognition capabilities using an advanced molecular dynamics (MD) simulation approach involving bias-exchange metadynamics and Markov State Models. The in-solution binding preferences of affinity tagged NFO4 (VYMNRKYYKCCK) to chlorinated (OTA) and non-chlorinated (OTB) analogues of ochratoxin were evaluated by appending hexa-histidine tags (6× His-tag) to the peptide's N-terminus (NterNFO4) or C-terminus (CterNFO4), respectively. The untagged NFO4 (NFO4), previously shown to bind with high affinity and selectivity to OTA, served as the control. Results indicate that the addition of site-specific 6× His-tags altered the peptide's native fold and the ochratoxin binding mechanism, with the influence of site-specific affinity tags being most evident on the peptide's interaction with OTA. The tags at the N-terminus of NFO4 preserved the native fold and actively contributed to the nonbonded interactions with OTA. In contrast, the tags at the C-terminus of NFO4 altered the native fold and were agnostic in its nonbonded interactions with OTA. The tags also increased the penalty associated with solvating the peptide-OTA complex. Interestingly, the tags did not significantly influence the nonbonded interactions or the penalty associated with solvating the peptide-OTB complex. Overall, the combined contributions of nonbonded interaction and solvation penalty were responsible for the retention of the native hapten recognition capabilities in NterNFO4 and compromised native recognition capabilities in CterNFO4. Advanced MD approaches can thus provide structural and energetic insights critical to evaluate the impact of site-specific tags and may aid in the selection and optimization of the binding preferences of a specific biosensing element.