An elevated serum phosphorus (P) has been anecdotally described in premature infants receiving human milk fortified with donor human milk-derived fortifier (HMDF). No studies have prospectively investigated serum P in premature infants receiving this fortification strategy. In this single center prospective observational cohort study, extremely premature infants ≤ 1250 grams (g) birth weight (BW) were fed an exclusive human milk-based diet receiving HMDF and serum P levels were obtained. We evaluated 93 infants with a mean gestational age of 27.5 ± 2.0 weeks (Mean ± SD) and BW of 904 ± 178 g. Seventeen infants (18.3%) had at least one high serum P level with a mean serum P of 9.2 ± 1.1 mg/dL occurring at 19 ± 11 days of life. For all infants, the highest serum P was inversely correlated to the day of life of the infant (p < 0.001, R2 = 0.175) and positively correlated with energy density of HMDF (p = 0.035). Serum P was not significantly related to gender, BW, gestational age, or days to full feeds. We conclude that the incidence of hyperphosphatemia was mild and transient in this population. The risk decreased with infant age and was unrelated to gender, BW, or ethnicity.