The effects of lactoferrin (LF) on the immune system have already been shown by many studies. Unfortunately, the current methods used to measure LF levels in milk do not permit the study of the genetic variability of lactoferrin or the performance of routine genetic evaluations. The first aim of this research was to derive a calibration equation permitting the prediction of LF in milk by mid-infrared spectrometry (MIR). The calibration with partial least squares on 69 samples showed a ratio of standard error of cross-validation to standard deviation equal to 1.98. Based on this value, the calibration equation was used to establish an LF indicator trait (predicted LF; pLF) on a large number of milk samples (n = 7,690). A subsequent study of its variability was conducted, which confirmed that stage of lactation and lactation number influence the overall pLF level. Small differences in mean pLF among 7 dairy breeds were also observed. The pLF content of Jersey milk was significantly higher than that in Holstein milk. Therefore, the choice of breed could change the expected LF level. Heritability estimated for pLF was 19.7%. The genetic and phenotypic correlations between somatic cell score and pLF were 0.04 and 0.26, respectively. As somatic cell score increases in presence of mastitis, this observation seems to indicate that pLF, or a function of observed pLF, compared with expected LF might have potential as an indicator of mastitis. The negative genetic correlation (-0.36) between milk yield and pLF could indicate an undesirable effect of selection for high milk production on the overall LF level.