This study investigated the expression profile of the Toll-like receptor 2 (TLR2) gene in goats across three reproductive groups – twin-bearing (n = 12), single-bearing (n = 10) and non-pregnant (n = 8) – using quantitative real-time PCR (qRT-PCR). Descriptive statistics revealed that twin-bearing goats were significantly heavier at service and kidding, with higher litter weights compared to other groups (p < 0.05). No breed-wise differences were observed, allowing pooled analysis for gene expression. Melt curve and amplification plot analyses confirmed primer specificity and efficient amplification. TLR2 expression was significantly upregulated in twin-bearing goats (ΔCt = −2.14 ± 0.89), with a mean 4.5-fold increase (p < 0.001) compared to non-pregnant controls, suggesting enhanced immune activation. Single-bearing goats showed negligible and non-significant changes in expression (ΔCt = −0.01 ± 1.45; p = 0.984). Statistical comparisons showed a significant difference in TLR2 expression between twin- and single-bearing groups (mean difference: 3.39; p < 0.001). These results reveals a significant association of twin pregnancies with elevated expression level of TLR2 gene in resource population. While the functional implications require further investigation, this upregulation may be associated with immune adaptations to increased gestational load. Overall, this study identifies TLR2 as a candidate gene expression marker strongly correlated with twin pregnancy in goats. Further validation through longitudinal studies on larger cohorts and investigation of tissue-specific expression is necessary to assess its potential predictive value for reproductive performance in small ruminants.