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Simple Summary: Salmonellosis is the second most reported gastrointestinal disorder in the European Union, resulting from the consumption of Salmonella-contaminated foods. Chickens are considered reservoirs of this pathogen among food-producing animals, for which hygienic and sanitary measures mitigate the risk to humans through the food chain. However, data about the presence of this pathogen in autochthonous Portuguese chickens or their by-products is scarce. In this context, the aim of this study was to conduct a preliminary investigation on the occurrence of Salmonella spp. in autochthonous Portuguese laying hens raised in a semi-extensive system for small-scale production. The screening revealed an absence of Salmonella spp. in all cloaca, eggshell, and litter material samples collected (n = 279) from the 31 selected flocks. Considering these results and the fact that Salmonella is still the leading cause of food-borne outbreaks, the risk posed by Portuguese autochthonous chicken breeds produced through alternative and extensive farming methods can be considered low. However, this risk should not be neglected and needs to be further investigated, using a larger sample size, to validate this trend. The sustainability of agroecological systems, biodiversity protection, animal welfare, and consumer demand for higher quality products from alternative and extensive farming methods have reinforced interest in local breeds that are well adapted to low-input environments. However, food safety needs to be safeguarded to reinforce consumer confidence. The aim of this study was to conduct a preliminary investigation on the occurrence of Salmonella spp. in eggshells, hen's cloaca, and litter materials from autochthonous Portuguese laying hens raised in a semi-extensive system for small-scale production. A total of 279 samples from 31 flocks belonging to 12 farms were obtained, with 63 samples from the "Preta Lusitânica" breed, and 72 samples each from the remaining autochthonous breeds, namely, "Branca", "Amarela", and "Pedrês Portuguesa". None (0%) of the samples analyzed were positive for Salmonella spp. To the best of our knowledge, these are the first results of Salmonella evaluation from hen's cloaca, eggshells, and litter materials in autochthonous Portuguese chickens, suggesting that a semi-extensive production system can contribute to better food security and a lower risk to public health and the environment. [ABSTRACT FROM AUTHOR]