The speed of development of biotechnology within agriculture, industry, and medicine has changed our lives, and we need a biotechnological literacy to understand it. This implies that teachers in primary schools need to be biotechnologically literate in order to educate future generations. The aim of this study was to investigate Swedish pre-service primary school teachers’ knowledge of and attitudes towards biotechnology and contextualize the results by comparing them with a corresponding group of Spanish teachers. Data was collected using the established questionnaire instrument Biotech XXI and analyzed statistically. The findings demonstrate that Swedish pre-service primary school teachers have knowledge gaps relating to the basic genetic concepts that underpin biotechnology, although they are aware of biotechnological applications. Their attitudes are quite positive towards biotechnological applications in health, but less so to buying and using genetically modified products. Higher levels of knowledge were correlated with more positive attitudes, indicating an attitudinal basis for expanding the knowledge of and teaching practices for biotechnology among primary teachers. The level of knowledge and attitudes in the Swedish sample were similar to those of the Spanish teachers, suggesting a similar situation may be prevalent in many countries. The results indicate a need to reconsider the science curricula within pre-service primary school teacher training programs in order to better prepare primary teachers for teaching biotechnological literacy.