Background: Vitamin B12 deficiency (B12D) is associated with multiple risk factors and comorbidities; however, there are no firm guidelines regarding screening for B12D in the population at risk. We aimed to identify the risk factors and comorbidities associated with B12D in an adult population. Methods: Retrospective review of adult patients who received medical care in our large urban tertiary healthcare system between 1/1/2011 to 12/31/2020. Variables were compared between the group who had B12D and those who did not (NoB12D). Results: Patients with B12D (n=2,666) were younger than the NoB12D group (n=2,334) (62.2±18.5 vs 76.5±7.6 years; P<0.001). There were significantly higher associations in B12D group compared to NoB12D group of female sex, recreational drug use, CHF, CVA, COPD, asthma, peripheral neuropathy (PN), subacute combined degeneration of spinal cord (SCD), dementia), mild cognitive impairment (MCI), GERD, gastritis, inflammatory bowel disease (IBD), small bowel resection (SBR), colorectal surgery (CRS), bariatric surgery (BS), depression, bipolar disorder, anxiety disorder, schizophrenia, anemia, use of NSAIDs, proton pump inhibitor (PPI), histamin-2 receptor antagonist (H2RA), and lithium. Compared to White race, Black and Hispanic races had higher odds of B12D (OR 1.43, 95% CI 1.02–2.01; P=0.040, and OR 3.15, 95% CI 2.23–4.45; P<0.001, respectively). Comorbidities with greater odds of B12D included COPD and asthma (OR 1.41, 95% CI 1.12–1.78; P=0.003), anemia (OR 253.72, 95% CI 164.66–390.93; P<0.001), PN and SCD (OR 2.78, 95% CI 2.16–3.58; P<0.001), dementia and MCI (OR 3.37, 95% CI 2.40–4.74; P<0.001), gastrointestinal disorders (GERD, gastritis, IBD, SBR, CRS and BS) (OR 2.38, 95% CI 1.95–2.90; P<0.001), PPI use (OR 2.81, 95% CI 1.88–4.20; P<0.001), and NSAID use (OR 1.40, 95% CI 1.08–1.81; P=0.011). Conclusion: Younger age, female sex, Black and Hispanic races, recreational drug use, CHF, CVA, COPD, asthma, PN, SCD, dementia, MCI, GERD, IBD, SBR, colorectal surgery, bariatric surgery, depression, bipolar disorder, anxiety disorder, schizophrenia, anemia, use of PPI, H2RA, NSAIDs and lithium are associated with B12D.