Indigenous chickens (Gallus domesticus) are the predominant poultry species kept under external systems in the rural and peri-urban poultry sector of Africa, which predispose them to various parasitic infections. The aim of the study was to utilize RNA-seq technology to characterize the village chicken transcriptome response to nematode infection and identify molecular mechanisms that underlie host resistance to gastro-intestinal nematodes. Village chickens representing two geographical regions of South Africa were studied. A survey analysis was conducted on 87 households from Limpopo (n = 39) and KwaZulu-Natal (n = 48) provinces to obtain data on the production system and associated gastrointestinal parasites. From 191 faecal samples collected, 15.73% and 42.16% were positive for gastrointestinal parasites in KZN and Limpopo provinces respectively, with A. galli the most prevalent. Small intestines from chickens infested by A. galli observed histopathological changes, including necrosis and hypertrophy of the villi. A total of 18 (Limpopo) and 22 (KZN) samples of A. galli collected from 144 chickens were sequenced. Haplotype diversity was smaller using cytochrome C oxidase 1 gene as marker, with most genetic variation occuring within populations compared to between populations. Gene expression analysis revealed more differentially expressed genes in the comparison of Limpopo-infected vs non-infected (277) as compared to KZN-infected vs non-infected (190). The results also indicated that the duodenum, jejunum and ileum displayed distinct transcriptome characteristics with differences in gene transcription. The study suggests significant small intestine section-specific differentialy expression patterns and support the hypothesis that different regions of the intestine are impacted differently during the nematode invation due to the different functions they play. Functional analysis revealed the biological processes involved in immune response that were significantly enriched, which included response to stimulus, immune system process, signalling andimmune response. Arachidonic acid metabolism and T cell receptor signaling pathway, were among the most significantly impacted pathways. Overall, this study confirms gastrointestinal parasites as a problem affecting village chickens of Limpopo and KZN provinces. Insight into the gene expression patterns would assist in understanding the host-parasite interactions during infection and direct decisions of disease control strategies.