Increasing global temperatures threaten rice production, a vital staple for over half the world’s population, particularly due to the vulnerability of seedlings to heat stress, which can reduce rice growth and yield. Weedy rice (Oryza spp.), an invasive weed in paddy fields, has demonstrated a degree of heat resistance. To better understand the mechanisms behind this tolerance, we analyzed genome-wide gene expression and adaptive strategies during the seedling stage under heat stress. Four weedy rice genotypes and two controls (MR219 and MU201) were subjected to heat treatment at 44 °C for 0, 2 and 4 h, followed by RNA isolation and RNA-Seq analysis. Results showed a decrease in the stress tolerance gene expression in heat-susceptible (HS) genotypes, particularly those associated with the oxidative phosphorylation (OXPHOS) pathway. Conversely, heat tolerant (HT) samples exhibited upregulated genes related to the endoplasmic reticulum pathways. Physiological assessments indicated high sensitivity to heat stress within the first four hours, with HS samples displaying significant cellular damage and increased ion leakage. In contrast, HT samples demonstrated better physiological characteristics and identified genes involved in damage repair. They also show stronger gene network connectivity, highlighting their critical coordination role in stress resilience. These findings reveal diverse mechanisms of heat stress response in weedy rice, providing insights for selecting weedy rice genotypes and targeting heat tolerance genes in rice breeding.