COVID-19 pandemic has shown the need to detect new pathogens quickly. Currently, attention has been paid to the recent epidemic of the Monkeypox virus (MpxV), the etiological agent of the zoonotic disease monkeypox. As happened with COVID-19, diagnosis is the priority in public health interventions. In MpxV infection, PCR is routinely used to detect the viruses. However, the utility of the method is dependent on designing PCR-efficient primers. Therefore, it is important to consider computational approaches that adopt various parameters, including thermodynamics, in order to offer increasingly efficient PCRs. We propose to determine the stability of primers for the specific detection of MpxV considering the thermodynamics of folding. For this, primers directed to specific genes were designed, classified, and reclassified. Furthermore, we propose to determine their theoretical aggregation in an aqueous model guided by molecular dynamics to offer optimal primers. A relationship was observed between the thermodynamic stability and the conformational fluctuations of the primers in an aqueous medium. The thermodynamic stability of the primers designed by standard methods could be discriminated by the prediction of DNA folding at a constant temperature. The variability observed in the primers after being reclassified shows an interaction between the thermodynamic stability and conformational fluctuations in solution, which could affect the efficiency of the PCR reactions. Therefore, under the conditions of this study, we propose to consider specific primers targeting the OPG191 gene of MpxV for experimental demonstrations since they present additional optimal characteristics in terms of thermal stability and dynamic.