Accurate padlock probe ligation is essential for all RCA-based diagnostics and methods. A short probe must circularize only when perfectly matched to its target template. This study investigated whether the nucleotide sequence composition at the ligation junction of padlock probes influences ligation success. The ten-nucleotide sequence at the ligation junction for 160 padlock probes, each scored from zero (low) to four (high) in their ligation efficiency, was analysed in software R. Each probe's ten-base ligation junction was converted into overall nucleotide percentages of fractions and position-specific one-hot features. Spearman's rank correlation (with Bonferroni correction) and position-wise chi-square and Fisher tests found no significant relationship between base composition and ligation score. The padlock probe and target template pairs were ligated using SplintR ligase, cleaned with exonucleases, and assayed by qPCR. Higher ligation scores consistently produced lower Cq values. Melting temperatures remained uniform, ranging from 84 °C to 86 °C, and were uncorrelated with the score. Together, these results demonstrate that the local sequence context at the ligation junction, whether analyzed as broad nucleotide fractions or fine-grained positional patterns, does not, by itself, influence ligation efficiency under optimized design conditions, such as probe backbone and arm length features. The empirical scoring of probe performance correlates with amplification behavior and remains essential for reliable assay design. The findings support the use of standardized ligation conditions and highlight the importance of experimental validation when developing padlock probe-based diagnostics.