Objectives: To determine prevalence of vitamin B12 and folate deficiency and associations with cognitive performance in participants recruited for the Cognitive Health in Ageing Register: Investigational, Observational, and Trial Studies in Dementia Research: Prospective Readiness cOhort Study (CHARIOTPRO) SubStudy (CPRO-SS). Design: Cross-sectional analysis of data collected in the screening phase for the CPRO-SS. Setting: Participants were recruited from the Chariot Register at Imperial College London comprising approximately 39,000 community dwelling volunteers. Participants: Community dwelling individuals aged 60-85 years with B vitamin biomarker measures available were included (n=1946). After medical history and other exclusions, 1347 cognitively healthy participants were included for analysis of cognitive data. Measurements: Cognitive status was assessed with the Repeatable Battery for Neuropsychological Status (RBANS). Assays included vitamin B12 and folate, followed by serum methylmalonic acid and homocysteine levels for those with low vitamin B12. Gender-specific linear regression analysis was performed for associations between cognition and biomarkers. Non-gender specific regression for groups graded by B vitamin deficiency severity were also performed. Results: Vitamin B12 deficiency (<148pmol/L) was found in 17.2% of individuals and folate deficiency (<10nmol/L) in 1% of our participants. Low vitamin B12 was associated with poorer memory (p<0.03) in men. A high BMI predicted poorer attention and visuospatial indices (p<0.05). A regression analysis by B12 level revealed associations with poorer attention (β -6.46; p=0.004) for the deficient group and with immediate memory (β -2.99; p=0.019) for those categorised as severely deficient. Conclusion: Older men and women are prone to vitamin B12 deficiency with associated subtle and different domain-specific disruptive effects in measures of memory and attention. Elevated homocysteine and methylmalonic acid contributed to poorer cognitive performance. Novel groups at particular risk of cognitive deficit were identified for future interventional studies in this field.