Background: Primary hyperparathyroidism (PHPT) with low preoperative vitamin D levels is thought to be related to 4-gland hyperplasia. We reviewed final parathyroid pathology in relationship to preoperative vitamin D levels. Methods: A retrospective review was performed for all PHPT patients undergoing parathyroidectomy from 2001 to 2019. Patient demographics, laboratory studies, and final pathology were reviewed. Results: 2230 patients were included in the analysis, 78.1% were female with a mean age of 59 years. Patients were stratified into 3 groups based on their preoperative 25-hydroxy vitamin D levels; “deficient” (<20 ng/mL) (n = 319), “insufficient” (20–30 ng/mL) (n = 1108), or “sufficient” (>30 ng/mL) (n = 803). Patients with deficient vitamin D had a higher frequency of single adenoma (71%) compared to sufficient vitamin D patients (62%) (p < 0.001) and fewer hyperplastic glands (19%) compared to sufficient vitamin D level patients (25%) (p < 0.001). Conclusions: Vitamin D deficiency is more strongly associated with single-gland parathyroid disease than 4-gland hyperplasia. Further investigation into the complex interplay between vitamin D levels and autonomous parathyroid function is warranted.