Italian rainbow trout (Oncorhynchus mykiss) farms are periodically affected by outbreaks of lactococcosis caused by Lactococcus garvieae, and rearing basins reveal simultaneously asymptomatic and symptomatic fish. The present study, as part of an “in field” sanitary survey, was designed to describe the expression of immune related genes in infected versus healthy fish, as well as to explore a Near Infrared Spectroscopy (NIR) as a novel approach to discriminate between them. Measurements/sampling were performed in a farm of Northern Italy and included two groups of fish (150–300 g), sharing the same basin: asymptomatic (n = 10) and symptomatic (n = 9). Fingerling (n = 5, 15 g) were also sampled from the farm internal hatchery and considered as L. garvieae-free reference group. NIR spectra were collected from the abdominal region of the fish through the SCiO molecular device. Spleen and head kidney were sampled for bacterial detection (PCR). Head kidney was also subjected to gene expression analysis (qPCR). Seven out of nine (7/9) symptomatic trout were L.garvieae positive (bacterial DNA) while all the asymptomatic (10/10) and fingerling (2/2) were L. garvieae negative. SCiO scanning provided two different spectral populations, almost coinciding with the two fish groups considered (asymptomatic and symptomatic). Among cytokines, IL-1β showed significant up-regulation in symptomatic group compared to asymptomatic group (P < 0.05) while IL-8 showed very high (P < 0.01) and high significant (P < 0.05) differences in its up-regulation in symptomatic group compared to asymptomatic group and fingerling, respectively. IL-10 showed a tendency of significant over-expressed in symptomatic fish compared to fingerling (P < 0.1). TCR-β showed significant (P < 0.05) down-regulation in symptomatic fish compared to asymptomatic ones and MHC-II resulted in down-regulation in a very significant (P < 0.01) manner in symptomatic trout compared to fingerling and asymptomatic. Concerning immunoglobulin genes expression, the level of mRNA transcript of IgT was significantly higher in symptomatic trout compared to fingerling (P < 0.05) and tended to be higher in asymptomatic trout compared to fingerling (P < 0.1). No significant differences were observed for IL-6, TNF-α, TLR5, MHC-I and IgM. In this study, a NIR based approach was used for the first time in the field of fish pathology/immunology with the aim of differentiating between healthy and diseased fish. Moreover, our results on immune related genes modulation highlighted how the outcome of a L. garvieae infection might be influenced by a specific gene expression pattern, which is crucial in the mode of action of trout immune system against L. garvieae.